Structural modification of an orally active thrombin inhibitor, LB30057: Replacement of the D-pocket-binding naphthyl moiety

Article abstract of DOI:10.1016/S0968-0896(98)00044-3

An amidrazonophenylalanine derivative LB30057 (2) was identified as a potent (K(i)=0.38nM), selective, and orally active thrombin inhibitor. As a continuation of studies into benzamidrazone-based thrombin inhibitors, we have structurally modified compound 2 by replacing the naphthyl group with a variety of hydrophobic moieties. This study led to discovery of several compounds with significantly enhanced potency in thrombin inhibition without sacrificing selectivity against trypsin and oral absorption. The highest activity was obtained with compound 23 (K(i)=0.045nM). Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00044-3

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 869±876
Structural Modi®cation of an Orally Active Thrombin
Inhibitor, LB30057: Replacement of the D-Pocket-binding
Naphthyl Moiety
Koo Lee,* Sang Yeul Hwang, Seongwon Hong, Chang Yong Hong,
Chang-Seok Lee, Youseung Shin, Sangsoo Kim, Mikyung Yun, Yung Joon Yoo,
Myunggyun Kang and Yeong Soo Oh
Biotech Research Institute, LG Chemical Ltd/Research Park, P.O. Box 61 Yu Sung Science Town, Taejon 305-380, Korea
Received 22 January 1998; accepted 12 February 1998
AbstractÐAn amidrazonophenylalanine derivative LB30057 (2) was identi®ed as a potent (Ki=0.38 nM), selective, and
orally active thrombin inhibitor. As a continuation of studies into benzamidrazone-based thrombin inhibitors, we have
structurally modi®ed compound 2 by replacing the naphthyl group with a variety of hydrophobic moieties. This study
led to discovery of several compounds with signi®cantly enhanced potency in thrombin inhibition without sacri®cing
selectivity against trypsin and oral absorption. The highest activity was obtained with compound 23 (Ki=0.045 nM).
# 1998 Elsevier Science Ltd. All rights reserved.
Introduction
Our work on thrombin inhibitors has been focused on a
novel `4-TAPAP-like'5 structure (e.g. 1) containing a
benzamidrazone residue at the P1 position because of its
advantage of excellent selectivity for thrombin over
trypsin.6 In our recent study on these benzamidrazone-
based compounds, the N,N-cyclopentylmethylamine
derivative LB30057 (2) was found to be optimal in the
set of amide derivatives, providing us with a potent
(Ki=0.38 nM), selective (Kitryp/Kithr=8658), and orally
bioavailable (58% in dogs) compound.7,8 In order to
further enhance the thrombin inhibitory potency of this
series, we have undertaken structure-based optimiza-
tions, focusing on the aryl moiety of the sulfonamide
function that binds in the hydrophobic distal (D-)
pocket of thrombin. In this paper, we report a study in
which the naphthyl group of sulfonamide 2 is replaced
with a variety of hydrophobic moieties, along with stu-
dies of the activity of these new compounds.
Thrombin is a trypsin-like serine protease that plays a
key role in the blood coagulation process by catalyzing
the conversion of ®brinogen to ®brin. This enzyme has
long been recognized as a central regulator in thrombosis
and hemostasis, and its inhibition has thus become a
major therapeutic target in the treatment of cardiovas-
cular diseases such as myocardial infarction, unstable
angina, deep vein thrombosis, and pulmonary embolism.1
Direct-acting and small molecule inhibitors of thrombin
are of considerable interest,2 and numerous inhibitors
possessing peptidic or non-peptidic structures have been
reported.3 However, they generally su€er from either a
lack of selectivity for thrombin against trypsin or poor
oral bioavailability. While some selective thrombin
inhibitors have been approved as injectable anti-
coagulants and are in late-stage clinical trials, it is clear
that next generation thrombin inhibitors must have
both oral bioavailability and selectivity in order to be
more clinically e€ective anticoagulants.4
Key words: Thrombin; inhibitor; oral; selective; amidrazone.
*Author for correspondence. Tel: (42) 866-2258; fax: (42) 861-
2566; e-mail: kleec@lgchem.co.kr
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00044-3

870
K. Lee et al./Bioorg. Med. Chem. 6 (1998) 869±876
Results and Discussion
mixture contribute less to the activity. Substitution of a
cyclohexyl group in this series, which was considered an
optimum ring size in modeling, also led to a potent
inhibitor in compound 22.
All compounds illustrated in Table 1 were assayed in
vitro for their thrombin inhibitory activity; results were
expressed as Ki determined using human a-thrombin.
The hydrophobic moieties incorporated ®rst in place of
2-naphthyl group in sulfonamide 2 were the 5-N,N-
dimethyl-1-naphthyl, 5-methoxy-1-naphthyl, 6-methoxy-
2-naphthyl, 7-methoxy-2-naphthyl, and 6,7-dimethoxy-
2-naphthyl groups. These moieties were studied by the
Mitsubishi group prior to their argatroban discovery,
and reported to enhance thrombin inhibitory activity
over the 2-naphthyl group in a series of thrombin inhi-
bitors, the Na-arylsulfonyl-substituted argininamide
derivatives.9 Therefore, we prepared compounds 3±7
and found that only 7 exhibited an increased thrombin
inhibitory activity (sixfold), while others su€ered a six-
to 28-fold decrease. The large activity di€erence between
the two similar compounds 5 and 7 can be explained by
inspecting our earlier X-ray crystal structure8 of com-
pound 2 bound to thrombin. This structure indicates
that there is some space available for substitution of a
small hydrophobic group at the 6-position of the naph-
thyl ring, thus accommodating the methoxy group,
while the 7-position is too close to the enzyme wall of
Trp215. The 6-ethoxy substituent (8) was also quite well
tolerated in this space, resulting in only slightly less
anity than the methoxy group. The unsubstituted
1-naphthyl group (9) showed a dramatic decrease in
anity.
Further molecular modeling studies concerning the
potent inhibitor 10 suggested that homologation of the
fused cyclohexenyl ring would lead to more ecient
space-®lling of the hydrophobic D-pocket. The 2-benzo-
cycloheptenyl compound 23 prepared in this regard
showed a three fold increase in inhibitory potency over
compound 10, which is in fact a signi®cant improvement
versus 2 (ca. ninefold). We turned to biaryl moieties10
and prepared biphenyl and pyridothienyl derivatives 24
and 25 primarily because their synthetic starting materi-
als (i.e. sulfonyl chlorides) were commercially available;
compounds of both synthons showed good binding
anity for thrombin.
The 2-anthracenyl derivative11 26 also exhibited strong
thrombin anity, while the dihydrophenanthrene deri-
vative 27 was slightly less active than 2. Compound 26,
however, su€ers from relatively poor water-solubility
compared to other derivatives despite its acid salt form.
We have also explored some non-aryl sulfonamide deri-
vatives such as styryl (28), benzyl (29), linear alkyl (30)
and bulky alkyl (31) groups. Although none of these
moieties o€ered any advantage in anity over the
2-naphthyl, it is notable that octyl and camphor deriva-
tives 30, 31 are only eight- and twofold less potent than
2, respectively, despite their lack of aryl functionality.
The 2-tetrahydronaphthyl group (10), another case of
better binding anity in the Mitsubishi's study,9 also
led to a three fold improvement in activity over com-
pound 2. This ®nding prompted us to investigate simple
alkyl-substituted benzenes as non-naphthalene aryl
moieties. Furthermore, a molecular modeling study had
suggested that a para-alkyl substituted phenyl moiety
would allow very favorable binding interactions in the
hydrophobic D-pocket, and that substituents such as an
isobutyl group would have a strong hydrophobic inter-
action with Leu99, Ile174 and Trp215 in the D-pocket
as shown in Figure 1. Accordingly, we prepared a series
of para-alkyl-substitued phenyl derivatives 12±18 by
increasing the chain length or bulk of the alkyl group. In
this series, the isobutyl-substituted compound 15,
indeed, showed the best thrombin inhibitory activity (three
fold more active than 2). Propyl-, butyl-, and neopentyl
substituents (13, 14, 17) were also well tolerated, leading
to reasonable binding anities. Additional substitution
of a methyl group at the meta-position to the sulfonyl
had little e€ect in increasing binding anity (19, 20)
even though the methyl group appeared to have an
additional hydrophobic interaction with the residues
Ile174 and Trp215. We assume that the isomers which have
the methyl group para to the sulfonyl in the inseparable
Compounds 7, 10, 15, 23, 24 and 26, which were found
to have excellent thrombin inhibitory potency, were
selected for further evaluation in which their selectivity
for thrombin compared to the related serine protease
trypsin (Table 2). Most of these compounds displayed
micromolar level of binding anity to trypsin, thus
providing excellent selectivity for thrombin. Preliminary
evaluation of the oral bioavailability of the selected
compounds was also performed. Compounds 7, 15 and
24 were found to have comparable pharmacokinetic
pro®les to that of 2 in rats,12 whereas compounds 10,
23 and 26 exhibited relatively low oral absorption
behavior. An in vivo ecacy study of these compounds
is currently underway; the results and details of the
pharmacokinetic study will be described elsewhere.
Synthesis
The target compounds listed in Table 1 were synthesized
as outlined in Scheme 1. Arylsulfonyl chlorides that
were not commercially available were prepared by two
di€erent approaches: chlorination of sulfonic acids or
their sodium salts (for compounds 4±8, 24, 26 and 27)
and direct chlorosulfonylation (for 10±23). Coupling of

K. Lee et al./Bioorg. Med. Chem. 6 (1998) 869±876
871
Table 1. Thrombin inhibitory activity of p-amidrazonophenyl alanine derivatives
Compd
R
Ki (nM) Compd
R
Ki (nM)

872
K. Lee et al./Bioorg. Med. Chem. 6 (1998) 869±876
Figure 1. Stereoview of 15 (yellow) modeled in the crystal structure of 2 (magenta)-thrombin complex.
Table 2. Trypsin inhibitory activity and selectivity ratios of p-
amidrazonophenyl alanine derivatives
as a major product from the corresponding 1-sub-
stituted regioisomer. However, asymmetric 1,2-di-
alkylbenzenes a€orded four regioisomeric sulfonamides
from which the 4- and 5-sulfonyl isomers were obtained
as an inseparable mixture. All of the target compounds
described here were obtained as pure acid salts by suc-
cessive puri®cation involving normal column chroma-
tography, HPLC and ion-exchange chromatography.
Compd
Ki (mM, trypsin)
Kitryp/Kithr
7
9.2
4.6
6.8
5.5
6.0
0.80
143750
38333
75555
122222
28571
8000
10
15
23
24
26
In summary, 30 di€erent hydrophobic moieties have
been investigated in place of the 2-naphthyl group of the
potent oral thrombin inhibitor 2, and the derivatives
were evaluated for thrombin inhibitory activity. Several
of these compounds turned out to be more potent than
compound 2 itself, with the best compound displaying
an order of magnitude enhancement in potency, as
well as being highly selective against trypsin. Further
structure±activity relationship studies for improving oral
bioavailability are currently under investigation;
the results of these studies will be published in due
course.
the sulfonyl chlorides with (S)-3-(4-cyanophenyl)-N-cyclo-
pentyl-N-methyl-2-(t-Boc-amino)-propionamide hydro-
chloride (33)6,7 gave intermediates of the general structure
34. These benzonitrile intermediates were smoothly
converted to benzamidrazones 3±31 in good yields by a
simple three step sequence involving sequential treatment
with hydrogen sul®de, methyl iodide and hydrazine. In
the case of monoalkyl benzenes, chlorosulfonylation
gave an inseparable mixture of para- and ortho-sub-
stituted sulfonyl chlorides in a 2:1 to 4:1 ratio in gen-
erally good yields. However, upon coupling with 33, a
mixture of two regioisomeric sulfonamides that were
separable by column chromatography was generated.
In the same manner, each of the 2-tetrahydronaphthyl
and 2-benzocycloheptenyl sulfonamides was separated
Experimental
The starting arylsulfonyl chlorides for the preparation
of compounds 3, 9, 12, 13, 14, 16, 18, 24, 25, 28, 29, 30
and 31 were purchased from Aldrich and Maybridge,

K. Lee et al./Bioorg. Med. Chem. 6 (1998) 869±876
873
Scheme 1. (a) N-Cyclopentyl-N-methylamine, EDC, HOBt, DMF; (b) AcCl, MeOH; (c) SOCl2, DMF; (d) HOSO2Cl, neat; (e) 33,
4-methylmorpholine, DMF; (f) i. H2S, pryidine, Et3N; ii. Mel, CH3CN; iii. NH2NH2.xH2O (80%), MeOH; (g) i. HPLC, H2O/MeOH;
ii. Dowex 1Â8-100 resin.
except biphenylsulfonyl chloride, which was purchased
from Fluka. Methoxy- and ethoxy-substituted naphtha-
lenesulfonyl chlorides for compounds 4±8 were pre-
pared from the corresponding hydroxy-substituted
naphthalenesulfonic acids and thionyl chloride accord-
ing to literature procedure.13 Arylsulfonyl chlorides for
compounds 26 and 27 were prepared from the corre-
sponding sulfonic acids which were commercially avail-
able (Sigma-Aldrich Rare Chemicals). The solvents
employed in these experiments were purchased and were
not puri®ed. NMR spectra were recorded on a Jeol
500 spectrometer; chemical shifts are reported in d
(ppm) relative to tetramethylsilane. Column chroma-
tography was carried out with Merck grade 60 silica gel
(230±400 mesh). Final puri®cation was achieved by
preparative HPLC on a Delta-Pak C18 column, 100-A
pore size, with tri¯uoroacetic acid (0.1%)-H2O-MeOH
solvent systems using various linear gradients. FABMS
and HR FABMS were obtained on a Jeol DX300 mass
spectrometer.
carbonylamino)propionic acid7,14 (7.0 g, 24.1 mmol) in
DMF (60 mL) was added EDC (5.08 g, 26.5 mmol) and
HOBt (3.58 g, 26.5 mmol), and the mixture was stirred
at rt for 7 h. A mixture of N-cyclopentyl-N-methylamine
hydrochloride7,15 (3.43 g, 25.3 mmol) and 4-methylmor-
pholine (4.0 mL, 50 mmol) in DMF (50 mL) was added,
and the mixture was stirred for another 9 h. After the
solvent was removed in vacuo, the residue was dissolved
in EtOAc (200 mL), washed with water, dried over
MgSO4, and concentrated in vacuo. Puri®cation by col-
umn chromatography (hexane: EtOAc, 3:7) a€orded
(S)-3-(4-amidrazonophenyl)-N-cyclopentyl-N-methyl-
2-(butyloxycarbonylamino)propionamide (32, 7.43 g,
83%): 1H NMR (CDCl3, rotamers) d 7.61 (m, 2H), 7.32
(m, 2H), 5.48 (m, 1H), 4.98, 4.90, 4.82 and 4.14 (4m,
2H), 2.79 and 2.64 (2s, 3H), 3.05 (m, 2H), 1.93-1.23 (m,
8H), 1.41 (s, 9H). FAB MS=372 (M++H).
This compound (7.43 g, 20 mmol) was dissolved in
MeOH (50 mL) and acetyl chloride (2.36 g, 30 mmol)
was added. After the mixture was stirred at rt for 5 h,
the solvent was removed in vacuo and the residue was
dried under high vacuum to produce compound 33
(5.85 g, 95%) as a white solid: 1H NMR (CD3OD,
(S)-3-(4-Amidrazonophenyl)-N-cyclopentyl-N-methyl-2-
aminopropionamide hydrochloride (33). To an ice-
cooled solution of (S)-3-(4-cyanophenyl)-2-(butyloxy-

874
K. Lee et al./Bioorg. Med. Chem. 6 (1998) 869±876
rotamers) d 7.77 (m, 2H), 7.50 (m, 2H), 4.82, 4.70 and
4.02 (3m, 2H), 3.22 (m, 2H), 2.80 and 2.61 (2s, 3H),
1.98-0.90 (m, 8H). FAB MS=272 (M++H). This pro-
duct was used directly without further puri®cation.
nophenyl)-N-cyclopentyl-N-methyl-2-(5,6,7,8-tetrahydro-
naphthalene-1-sulfonyl)propionamide hydrochloride (11).
To an ice-cooled ¯ask containing chlorosulfonic acid
(2.3 mL, 34.6 mmol) was added 1,2,3,4-tetrahydro-
naphthalene (2.0 mL, 14.2 mmol) dropwise over 10 min.
The resulting mixture was stirred for 3 h at rt, poured
into ice water and extracted with EtOAc (20 mLÂ3).
The combined extracts were washed with water
(20 mLÂ2), dried over MgSO4 and concentrated in vacuo
to give a mixture of 2- and 1±5,6,7,8-tetrahydronaph-
thalenesulfonyl chlorides (1.3 g, 96%) in a ca. 3:1 ratio
as determined by 1H NMR. 1H NMR (CDCl3) major
regioisomer, d 7.74 (d, 1H), 7.40 (d, 1H), 7.27 (d, 1H),
2.89 (s, 4H), 1.85 (s, 4H); minor regioisomer 7.91 (d, 1H),
7.30 (t, 1H), 7.10 (m, 1H), 2.80 (m, 4H), 1.83 (m, 4H).
Synthesis of (S)-3-(4-amidrazonophenyl)-N-cyclopentyl-
N-methyl-2-dansylpropionamide hydrochloride (3). To a
solution of 33 (2.0 g, 6.5 mmol) in DMF (50 mL) was
added 4-methylmorpholine (2.14 mL, 19.5 mmol) and
dansyl chloride (1.75 g, 6.5 mmol), and the mixture was
stirred for 1 h at rt. After the resulting solution was
concentrated in vacuo and the residue dissolved in
CH2Cl2, the solution was washed with water. The
organic layer was dried (MgSO4), concentrated, and
puri®ed by column chromatography (hexane: EtOAc,
7:3) to provide the coupling product, (S)-3-(4-cyano-
phenyl)-N-cyclopentyl-N-methyl-2-dansylpropionamide,
The crude sulfonyl chloride mixture (1.5 g, 6.52 mmol)
was added to a solution of 33 (2.0 g, 6.5 mmol) and 4-
methylmorpholine (2.14 mL, 19.5 mmol) in DMF
(10 mL) and the mixture was stirred for 1 h at rt. After
standard workup, the regioisomeric coupling products
were separated by column chromatography (hexane:
EtOAc, 3:2). For major product (1.40 g, 46%), (S)-3-(4-
cyanophenyl)-N-cyclopentyl-N-methyl-2-(5,6,7,8-tetra-
hydronaphthalene-2-sulfonyl)propionamide: 1H NMR
(CDCl3, rotamers) d 7.55 (m, 2H), 7.45 (m, 1H), 7.26
(m, 3H), 7.10 (m, 1H), 5.72 (d, 1H), 4.56, 4.49, 4.29 and
3.81 (4m, 2H), 2.97 (m, 2H), 2.80 (bs, 4H), 2.60 and 2.34
(2s, 3H), 1.80±0.90 (m, 12H); FAB MS=466 (M++H).
For minor product (0.46 g, 15%), (S)-3-(4-cyanophenyl)-
N-cyclopentyl-N-methyl-2-(5,6,7,8-tetrahydronaphthalene-
1-sulfonyl)propionamide: 1H NMR (CDCl3, rotamers)
d 7.71 (m, 1H), 7.52 (m, 2H), 7.18 (m, 3H), 7.10 (m, 1H),
5.78 (d, 1H), 4.57, 4.48, 4.28 and 3.78 (4m, 2H), 3.20±
2.80 (m, 6H), 2.60 and 2.26 (2s, 3H), 1.90±0.90 (m,
12H); FABMS=466 (M++H).
1
as a white solid (3.05 g, 93%): H NMR (CDCl3, rota-
mers) d 8.54 (m, 1H), 8.16 (m, 2H), 7.71±7.04 (m, 7H),
6.00 (d, 1H), 4.53, 4.40, 4.34 and 3.82 (4m, 2H), 2.90 (m,
8H), 2.50 and 2.26 (2s, 3H), 1.80±0.88 (m, 8H). FAB
MS=505 (M++H).
A
solution of the coupling compound (0.347 g,
0.69 mmol) in pyridine (15 mL) was saturated with gas-
eous H2S. After the mixture was allowed to stand for
2 d, the solvent was removed in vacuo to obtain the
thioamide as a yellow solid. To this material was added
CH3CN (15 mL) and iodomethane (0.13 mL, 2.09 mmol),
and the mixture was heated at re¯ux for 1 h. After the
solvent was evaporated in vacuo, the resulting methyl-
thioamidate was dissolved in absolute MeOH. To this
solution was added portionwise 80% hydrazine hydrate
(0.12 mL, 1.98 mmol) over 10 min, and the mixture was
stirred at rt. The solution was concentrated and the
residue puri®ed by column chromatography using 10%
MeOH in CHCl3 to give the title compound which was
further puri®ed by preparative HPLC (tri¯uoroacetic
acid (0.1%)-H2O-MeOH gradient). The pure fractions
were passed through an ion-exchange resin column
(Dowex 1Â8-100) and then lyophilized to give a white
Each coupling product was sequentially treated with
hydrogen sul®de, methyl iodide, and hydrazine to give
an amidrazone as described for the preparation of
compound 3. For compound 10 (1.07 g, 67%), 1H NMR
(CD3OD, rotamers) d 7.65 (m, 2H), 7.42 (m, 4H), 7.15
(m, 1H), 4.55, 4.40 and 4.04 (3m, 2H), 3.05 and 2.90
(2m, 2H), 2.81 (m, 4H), 2.60 and 2.51 (2s, 3H), 1.90±1.00
(m, 12H). HR FAB MS calcd for C26H36N5O3S
498.2538 (M++H), found 498.2534. For compound 11
1
solid 3 (0.24 g, 65%) as a HCl salt: H NMR (CD3OD,
rotamers) d 8.50 (m, 2H), 8.21 (m, 1H), 7.68±7.59 (m,
5H), 7.32 (m, 2H), 4.67, 4.43, 4.22 and 3.98 (4m, 2H),
3.11 (s, 3H), 3.08 (s, 3H), 3.00 and 2.87 (2m, 2H), 2.46
and 2.38 (2s, 3H), 1.76±0.90 (m, 8H). HR FABMS
calcd for C28H37N6O3S 537.2647 (M++H), found
537.2658.
1
(0.38 g, 72%), H NMR (CD3OD, rotamers) d 7.70 (m,
1H), 7.60 (m, 2H), 7.36 (m, 2H), 7.25 (m, 1H), 7.16 (m,
1H), 4.50, 4.38, 4.34 and 3.91 (4m, 2H), 3.10±2.90 (m,
4H), 2.78 (m, 2H), 2.45 and 2.39 (2s, 3H), 1.85±0.97 (m,
12H). HR FABMS calcd for C26H36N5O3S 498.2538
(M++H), found 498.2541.
In similar procedure to the synthesis of 3, compounds
4±9, 12±14, 16, 19 and 22±31 were prepared from the
corresponding sulfonyl chlorides.
Synthesis of (S)-3-(4-amidrazonophenyl)-N-cyclopentyl-
N-methyl-2-(5,6,7,8-tetrahydronaphthalene-2-sulfonyl)-
propionamide hydrochloride (10) and (S)-3-(4-amidrazo-
Compounds 15, 11, 17, 18, 19 and 20±23 were prepared
from the corresponding alkylbenzenes in a similar man-
ner to the synthesis of 10.

K. Lee et al./Bioorg. Med. Chem. 6 (1998) 869±876
875
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The activity of thrombin was measured spectro-
photometrically using tosyl-Gly-Pro-Arg-p-nitroanilide
acetate (Chromozyme TH, Boehringer Mannheim) as a
substrate. Thrombin used in the tests was prepared from
human plasma according to the protocol of Ngai and
Chang.16 Each compound was dissolved in DMSO to
make a 1 mM stock solution and dilutions were made
thereof with assay bu€er (0.1 M Tris±HCl, 0.15 M
NaCl, 0.1% polyethylene glycol 8000, pH 7.8). Di€erent
concentrations of inhibitor were mixed with 0.3 NIH
units of thrombin in 0.8 mL of the bu€er. The mixture
was incubated for 10 min at rt before adding 0.2 mL of
the substrate to a ®nal concentration of 20 mM. The
release of p-nitroaniline by hydrolysis of the substrate
was monitored for 5 min by measuring the increase in
optical density at 381 nm with a UV2100S spectrometer
(Shimadzu). A graph for the reciprocal value of initial
velocity to the inhibitor concentration was derived from
progress curves by ®tting the data using a linear regres-
sion program. The inhibition constants (Ki values) were
then obtained from the Dixon plot equation.17 Under
these conditions, the Km value for the substrate hydro-
lysis was 5.2 mM as determined from a non-linear
regression analysis of initial rate assuming Michaelis±
Menten kinetics.
Inhibition constants for trypsin were determined as
described above using 1.2 mg/mL of bovine pancreatic
trypsin (Sigma) and 20 mM of N-benzoyl-Val-Gly-Arg-
p-nitroanilide hydrochloride (B-4758, Sigma). The Km
of the substrate was 10.5 mM.
4. Kimball, S. D. Blood Coag. Fibrinol., 1995, 6, 511.
5. Na-(4-toluenesufonyl)-p-amidinophenylalanyl-piperidine: (a)
Markwardt, F.; Wagner, G; Sturzebecher, J.; Walsmann, P.
Thromb. Res. 1980, 17, 425. (b) Turk, D.; Sturzebecher, J.;
Bode, W. FEBS Lett. 1991, 287, 133.
Acknowledgements
We wish to thank Drs J. M. Cho, Y. Z. Kim, and I. C.
Kim for their support.
6. Kim, S.; Hwang, S. Y.; Kim, Y. K.; Yun, M. K.; Oh, Y. S.
Bioorg. Med. Chem. Lett. 1997, 7, 769.
7. Oh, Y. S.; Kim, S. S.; Hwang, S. Y.; Yun, M. K.; Hwang, S.
R.; Hong, S. W.; Lee, Y. H.; Jeong, Y. N.; Lee, K.; Shin, Y. S.
European Patent Application EP739886-A2, 1996; Chem.
Abstr. 1996. 126, 47554.
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