Selective boron-containing thrombin inhibitors - X-ray analysis reveals surprising binding mode

Article abstract of DOI:10.1016/S0968-0896(00)00147-4

Based on the structural comparison of the S1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesized as inhibi

Full text of DOI:10.1016/S0968-0896(00)00147-4

Bioorganic & Medicinal Chemistry 8 (2000) 2291±2303
Selective Boron-Containing Thrombin InhibitorsÐX-ray
Analysis Reveals Surprising Binding Mode
a,
a
b
a
Anette von Matt, * Claus Ehrhardt, Peter Burkhard, Rainer Metternich,
c
a,,
Malcolm Walkinshaw and Carlo Tapparelli
aNovartis Pharma AG, CH-4002 Basel, Switzerland
Biozentrum UniversitaÈt Basel, CH-4056 Basel, Switzerland
b
cUniversity of Edinburgh, Edinburgh EH9 3RJ, UK
Received 27 March 2000; accepted 31 May 2000
AbstractÐBased on the structural comparison of the S1 pocket in di€erent trypsin-like serine proteases, a series of Boc-d-tri-
methylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being di€erent amino boronic acids, have been synthesized
as inhibitors of thrombin. Among the novel compounds, a number of derivatives were synthesized which appeared to have side-
chain variants too big to ®t into the S1 pocket. Nevertheless, these compounds inhibited thrombin in the nM range. The X-ray
structure of one of these inhibitors bound to the active side of thrombin reveals that a new binding mode is responsible for these
surprising results. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Because of the important role of thrombin in the coagu-
lation mechanism, the enzyme has become a key target
for the treatment of thromboembolic diseases.3
In a number of important cardiovascular diseases such as
myocardial infarction, unstable angina, deep vein
thrombosis, pulmonary embolism, and ischemic stroke,
a causative factor is intravascular clot formation. A
blood clot is formed by activation of the coagulation
cascade through the intrinsic (`contact activation') or
the extrinsic (activation by exposure of plasma to a
nonendothelial surface or damage to vessel wall and
tissue factor release) coagulation pathways.
Up to now only two anticoagulant strategies have been
used clinically for treatment of acute and chronic
thrombosis: the use of heparins which act by activation of
endogenous plasma proteins that inhibit thrombin and
other proteases of the coagulation cascade, and the use of
coumarins which inhibit the hepatic synthesis of vitamin
K-dependent proteins including thrombin. Because of the
indirect and non-selective mechanism of these anti-
coagulants, careful monitoring is necessary and side
e€ects often can be observed.4 Therefore, much e€ort
has been invested to ®nd novel classes of low molecular
weight, direct acting inhibitors of thrombin, which are
safe and selective.6
The enzyme which plays a pivotal role in clot formation
is thrombin, a trypsin-like serine protease. Thrombin is
the last enzyme in the coagulation cascade. It converts
soluble ®brinogen to ®brin which then can polymerize
to form a haemostatic plug.1
,5
In addition to ®brinogen cleavage and platelet activation,
thrombin exerts a positive feedback to its own production
by activating coagulation factors V and VIII. It also acti-
vates factor XIII which crosslinks and stabilizes the ®brin
polymers and stimulates platelet secretion and aggregation
through a proteolytic activation of the thrombin receptor
There are two principal classes of low molecular weight
active site directed thrombin inhibitors: the ®rst consists
of inhibitors which do not form a covalent bond to the
catalytic Ser 195 but bind non-covalently through
hydrogen-bonding, hydrophobic, and electrostatic inter-
actions within the S1±S3 speci®city pockets. Examples of
2
7
8
on the platelets.
this class of compounds are Argatroban, NAPAP, Ino-
9
10
11
gatran, Melagatran, Napsagatran, and several other
non transition state molecules.1
2�21
The second class of
inhibitors form a covalent bond between the nucleophilic
Ser 195 of the enzyme and an electrophilic center on the
*Corresponding author. Tel.: +41-61-324-2096; fax: +41-61-324-3036;
e-mail: anette.von_matt@pharma.novartis.com
,
Deceased.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00147-4

2
292
A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2
2
inhibitor. Chloromethylketones like PPACK, C-term-
2
All compounds are peptide boronates of the type PG-d-
TMSal-Pro-boroLys(X) pinanediol (PG=protecting
group) with constant building blocks in P2 and P3 and
variations on the amino function of the borolysine side-
chain (X).
3
24
25
inal aldehydes, ¯uoromethylketones, a-ketoesters or
amides, phosphonates and phosphonites, and boronic
2
6
27
2
8�31
are examples of this second class.
acids
Many of these thrombin inhibitors have a basic P1 side-
chain in order to form a salt bridge to Asp 189 at the bot-
tom of the S1 pocket and thereby increase the inhibitory
anity for thrombin. However, it has been shown that a
basic P1 side-chain is not a necessary prerequisite to
d-Trimethylsilyl alanine (d-TMSal) in P3 was used as
bioisosteric replacement of d-phenylalanine to reduce
the metabolic instability of our molecules.41
obtain active thrombin inhibitors.3
2�34
Despite the fact that most of the synthesized compounds
have the Boc protecting group at the N-terminus, the X-
ray structure determination has been done with the Cbz
protected formamide derivative 18, since this compound
was a candidate for clinical development. However, the
N-terminal hydrophobic protecting group should not
have a great in¯uence on the binding mode of these
compounds, because the activities of the Boc-protected
compound 9 and the Z-protected compound 18 vary by
a factor of 2±3 only (see Table 2).
In a recent paper,35 we have described selective boron
containing thrombin inhibitors with non-basic residues
in P1, which showed a higher selectivity for thrombin over
trypsin and plasmin. Most of the new P1 side-chains are
closely related to those of Lys or Arg.
Continuing our search for selective inhibitors of thrombin,
we have now extended the P1 variations to side-chains
containing butyl formamides, thioformamides, alkyl
amides and ureas. According to molecular modeling
studies, these side-chains seem to be too big to ®t into
the S1 pocket of thrombin. Nevertheless, these com-
The same similarity of activities can be observed for
compound 8 with a Boc and compound 20 with an iso-
butylcarbonyl moiety at the N-terminus (variations by a
factor of 1±2). Here synthetic reasons are responsible
for the di€erent N-termini. The Boc protecting group is
not stable under the reaction conditions we used to
prepare the free boronic acid while the isobutylcarbonyl
moiety is compatible with these conditions.
pounds show K values for thrombin in the pM±nM
i
range. A novel binding mode which could explain the
high potency of these compounds has been found by
X-ray analysis.
Inhibitor Design
Chemistry
In order to gain selectivity for thrombin over trypsin
and plasmin, the S1 pocket of these serine proteases is
All thrombin inhibitors described in this paper have been
synthesized starting from the common intermediate 7
(see Scheme 2). The strategy to access 7 and all other
compounds described in this report was based on a
3
6
an attractive target. This region is quite conserved in
all three proteases but there are still small di€erences
which can be used to discriminate one serine protease
3
7
35
from the others.
previous report from these laboratories.
It has been demonstrated that a neutral P1 side-chain in
peptide boronic acid inhibitors can improve the selectivity
for thrombin over trypsin and plasmin quite
The ®rst building block needed was Boc-d-TMSal-Pro-
ONSu 3. It was prepared by coupling of proline to (S)-N-
(t-butoxycarbonyl)-b-(trimethylsilyl)alanine 1 (=Boc-d-
TMSal-OH) and subsequent activation with N-hydroxy
succinimide (Scheme 1). Boc-d-TMSal-OH 1 was
obtained using the Schoellkopf procedure as described in
the literature.41
3
5,38�40
dramatically.
From a synthetic point of view, a
very ecient pathway to neutral side-chains is derivati-
zation of the side-chain amino function of borolysine. In
one step, amide or urea derivatives can be synthesized.
We assumed that there is a certain size and volume of the
side-chain which is optimal for obtaining selectivity
without losing too much activity. Therefore, a number of
amides and ureas with di€erent demand on space were
synthesized. As the smallest representatives, formamide
and thioformamide derivatives were prepared.
The aminoboronic acid derivative which was needed for
the coupling to 3 was synthesized using a procedure
developed by Matteson.4
2�44
4-Bromo-1-propene was
treated with catecholborane and the crude product was
reacted with (+)-pinanediol to give the borane pinanediol
ester 4. Due to the chiral auxiliary, the `CHCl' insertion
Scheme 1.

A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2293
Scheme 2.
generated a single diastereomer of the chlorosubstituted
homologue 5. The reaction did not go to completion but
the unreacted starting material could be removed by
chromatography after the subsequent one-pot reaction:
substitution of Cl by N(SiMe ) , acidic hydrolysis and
protecting group with the isobutylcarbonyl moiety by
treatment with acetic acid/concd. HCl followed by
acylation with isobutyl acid chloride. The resulting
compound 19 was either hydrogenated to give the
pinanediol derivative 20, or treated with BCl and then
3
3
2
coupling with the activated dipeptide 3. The replace-
ment of Cl by N(SiMe ) led to inversion of the con-
hydrogenated which yielded the corresponding free
boronic acid 21 (Scheme 5).
3
2
®guration in the product. In the next step, Br was
exchanged by N and the common intermediate 7 could
be obtained (Scheme 2).
3
Biology
Hydrogenation of the azido group of 7 catalyzed by
palladium on carbon led to the borolysine derivative
All biological results are summarized in Table 2.
2
9
8
further derivatized by reaction either with an anhydride
.
The free amino function of this compound could be
Compounds with a lysine side-chain like 8, 20 and 21
were highly active thrombin inhibitors. As can be seen
with 20 and 21, there was no di€erence in the activity
for thrombin and the selectivity over plasmin and trypsin,
regardless of whether the boronic ester was hydrolyzed
or not. This indicates that pinanediol was removed
before the compounds bound to thrombin.45 The X-ray
crystal structure of 18 supported this hypothesis.
Although the pinanediol ester derivative of 18 has been
used for crystallization, no pinanediol could be
observed in the X-ray structure.
(
!9), acid chlorides (!10, 11, 12, 13), isocyanates
!14, 15), or potassium cyanate (!16) (Scheme 3;
(
Table 1).
The thioformamide derivative 17 could be obtained by
treatment of the corresponding formamide 9 with Law-
esson's reagent. Removal of the Boc protecting group
from 9 and reaction with benzyl chloroformate led to
compound 18 (Scheme 4).
The Boc-protecting group was not stable under the
conditions used to remove the pinanediol group. In
order to prepare the free boronic acid, it was replaced
by an isobutylcarbonyl capping group. Neither the N-
terminal unprotected free boronic acid nor the free
boronic acid with the lysine-side chain were stable and
thus not useful as intermediates. Therefore, we started
with the azido compound 7 and replaced the Boc
i
Table 2. Dissociation constants (K ) of the inhibitors were assessed in
a chromogenic assay with human thrombin, human plasmin and bovine
pancreatic trypsin. The selectivity for the inhibition for thrombin over
plasmin or over trypsin was compared on the basis of the ratios Ki,PLAS
/
Ki,THR or Ki,TRY/Ki,THR (in parenthesis). Mean values (n=3)
K
i
(nM)
No.
Thrombin
Plasmin
Trypsin
8
9
1
11
1
13
14
15
16
17
18
20
21
0.039
5.57
0.073
0.056
0.062
87.9
5.4
3.8
6.6
0.2
3.51(90)
8250(1530)
4.4(60)
8.4(150)
6.4(103)
3983(45)
8024(1486)
2597(683)
2155(327)
2795(13 975)
2493(993)
2.5(92)
0.39(10)
202(36)
1.0(14)
0.9(16)
0.8(13)
1860(21)
1447(268)
502(1325)
313.7(48)
63(315)
Table 1. Reagents and yields for Scheme 3
0
No.
R
Reagent
Yield (%)
2
9
H
Me
Et
i-Pr
NMe
MeC(O)OC(O)H
MeC(O)Cl
EtC(O)Cl
90
97
95
94
42
94
87
67
10
11
12
13
14
15
16
i-PrC(O)Cl
Me NC(O)Cl
2
2
NHMe
NHEt
NH
MeNCO
EtNCO
KOCN
2.51
0.027
0.054
105(42)
0.18(6.7)
0.18(3.3)
2
1.5(28)

2
294
A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
Scheme 3.
Scheme 4.
Di€erent N-terminal groups (Boc for 8 and isobutylcar-
bonyl for 20 and 21) seemed to have no big in¯uence on
the inhibitory activity. The same observation could be
made for the formamide compounds 9 and 18 with Boc
and Cbz protecting groups, respectively.
The same independence from substituent size was found
for the urea derivatives 14, 15 and 16. They were generally
less active than the alkyl derivatives 10±12 but again all
three compounds had quite comparable K values. Only
i
13, with two additional substituents on the nitrogen,
was less active. It could be that either the size of the two
methyl groups causes severe steric hindrance or the NH
is necessary for binding.
The formamide side-chain a€orded lower activity for
thrombin but higher selectivity over plasmin and
trypsin. Replacement of oxygen by sulfur leading to
thioformamide derivative 17 gave both higher activity
and selectivity.
X-ray Crystallographic Structure of 21 Bound to
Thrombin
A very surprising result was observed for the alkylcar-
bonyl derivatives 10, 11 and 12. Despite the much bigger
substituents the activity was comparable to the activity
of the lysine compound 8. There was no di€erence in
activity whether the alkyl group was methyl, ethyl or
isopropyl.
To gain further insight into the binding mode of the inhi-
bitors under investigation the X-ray structure of 21 bound
to human a-thrombin has been determined. The resolu-
Ê
tion was 2.3 A and the ®nal R-factor 19.1% (Table 4; for
crystallographic details, see Experimental).

A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2295
Scheme 5.
The inhibitor is bound to the protein by a covalent linkage
between the boronic acid and Ser 195 of thrombin. The
boron atom shows a tetrahedral coordination, with cova-
lent bonds to the Ca-carbon, to the two oxygen atoms
of the boronic acid and also to the Og oxygen atom of
Ser 195 (Fig. 1A). The NE2 nitrogen of His 57 forms a
Finally, the N-terminal portion of the ligand forms a
short anti parallel b-sheet with residue Gly 216 of the
protein (Fig. 1A; Table 3).
X-ray Crystallographic Structure of 18 Bound to
Thrombin
hydrogen bond to one oxygen atom of the boronic acid
Ê
2.97 A) which in turn forms a hydrogen bond to a
(
water molecule (Table 3).
To further elucidate the protein±ligand interactions in
the S1 pocket-compound 18 with a modi®ed P1 side-
chain complexed to human a-thrombin has been
determined by X-ray crystallography. The resolution
The lysine side-chain extends as expected into the S1
pocket (Fig. 1A). Its positively charged amino group
forms a salt-bridge to the side chain of Asp 189 and two
hydrogen bonds to the carbonyl oxygen atoms of residues
Gly 219 and Ala 190. There is an additional hydrogen
bond to a water molecule which is buried in the S1
pocket (Table 3).
Ê
was 2.3 A and the ®nal R-factor 17.4% (Table 4; for
crystallographic details, see Experimental).
Apart from the P1 residue, the structure of 18 bound to
thrombin is very similar to that of 21 despite the di€erent
N-terminal protecting groups (Fig. 1B; Table 3). The P1
side-chain, however, shows a new binding mode. It does
not contact Asp 189 but leaves the S1 pocket after the
Cd so that the terminal formamide can form two
hydrogen bonds with Gly 219 in a cis-conformation
(Fig. 1B; Table 3).
Table 3. Interatomic distances
Ê
Distance (A)
Protein
Ligand
2
1
There is no evidence for the pinanediol in the X-ray
structure. Boronic acids, even introduced as their corre-
sponding esters, form complexes with the enzyme only
as the free boronic acids. This is due to rapid equilibration
between the boronic acid ester and the free boronic acid.
There is also no evidence in the literature that the presence
Gly 219 O
Ala 190 O
Asp 189 Od2
Wat 2
Gly 193 N
Ser 195 N
Wat 1
His 57 NE2
Gly 216 O
Gly 216 N
boroLys N
boroLys N
boroLys N
boroLys N
boroLys O 1
boroLys O 1
boroLys O 2
boroLys O 2
TMSal N
z
z
z
z
2.93
2.75
3.16
2.81
2.88
2.99
2.31
2.97
3.27
3.06
4
5
TMSal O
Table 4. Crystallographic data statistics
1
8
1
8
21
2.3
19.1
23.5
a
Gly 219 N
Gly 219 O
Gly 193 N
Ser 195 N
Wat 1
His 57 NE2
Gly 216 O
Gly 216 N
Lym CHO
a
3.29
2.91
3.09
2.75
2.85
2.88
2.77
3.16
Lym N
z
Lym O 1
Ê
Resolution (A)
2.3
a
R
cryst (%)
free (%)
17.4
20.8
a
Lym O 1
a
R
rms bond dev (A)
Lym O 2
a
Ê
0.013
1.66
15 252
93.1
4.6
0.013
1.72
13 771
84.1
4.0
ꢀ
Lym O 2
TMSal N
TMSal O
rms angle dev ( )
Number of re¯ections
Completeness (%)
Free set (%)
aLym=boroformyl lysine.

2
296
A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
The comparison of the X-ray structures of 21 and 18
gives a simple explanation: while 21 forms a `traditional'
salt bridge to Asp 189 in S1 of thrombin with its Lys-
side-chain,3
0,45
compound 18 shows a new binding mode.
The side-chain enters the S1 pocket, but in a kind of a U-
turn leaves it again to form two new hydrogen bonds
between the formamide and the backbone of Gly 219.
One could argue that the new binding mode is due to
the di€erent N-terminal groups or the presence/absence
of pinanediol during crystallization. But all published
X-ray structures of boronic acid derivatives in complex
with thrombin bind in the `traditional' way irrespective
of their N- or C-terminal groups.3
9,40,45,46
Because of the loss of the strong salt bridge to Asp 189,
the activity is reduced towards all three enzymes. How-
ever, the loss of potency is much stronger in the case of
plasmin and trypsin, since the latter enzymes have a Ser
in position 190 compared to an Ala in thrombin so that
an additional hydrogen bond can be formed to a Lys
side-chain, as is evident from several trypsin/inhibitor-
complexes3
0,39,45
(Fig. 1C). Based on this argumenta-
tion, the selectivity pattern of all compounds under
investigation should be similar which is in contrast to
the results shown in Table 2. If one assumed a similar
binding conformation for all inhibitors in all enzymes,
there are two other e€ects that have to be taken into
account. The contacts of the P1 side-chains with the
protein backbone around position 217 (Fig. 1C) and the
interaction of the inhibitors with their own hydrophobic
protecting groups could also play a role for enzyme
selectivity.
Figure 1. X-ray structures of human a-thrombin complexed with the
inhibitors 18 and 21. (A) The complex with the inhibitor 21.; (B) the
complex with the inhibitor 18. (C) Overlay of the structures of
thrombin and trypsin complexed with inhibitor 21 and mung bean
inhibitor, respectively. Carbon atoms are coloured in yellow for the
protein atoms of thrombin, in cyan for the inhibitor 21 and in magenta
for the lysine side-chain of the mung bean inhibitor and the residues
Compounds 10, 11 and 12 with an alkylamide in P1
show very similar data compared to 8. One could think of
a hydrolysis of the Lys side-chain amide bond under assay
conditions, which would transform these compounds
back to 8. However, this amide bond is quite stable even
under more drastic conditions.
1
90, 218 and 219 of trypsin. The Bor atom is coloured in orange and
the Silicium atom is coloured in magenta. In trypsin, the residue
Ser190 makes a direct hydrogen bond to the side-chain nitrogen of the
lysine (magenta), while in thrombin there is a water molecule hydrogen
bonded to this nitrogen.
Probably the amide is in a trans-conformation in these
compounds, and the additional hydrophobic group can
now, in contrast to 9 or 18, stabilize the bound con-
formation by non-polar interactions with the Boc group,
thus compensating for the loss of the salt bridge. Since
this stabilization is possible in all enzymes the selectivity
compared to 8 does not change.
or absence of pinanediol during crystallization has any
in¯uence on the binding mode in the S1 pocket.
45
Discussion
In our previous paper,35 we could demonstrate how the
selectivity of tripeptide±boronic esters can be in¯uenced
by variations in P1, making use of the more hydrophobic
nature of the S1 pocket in thrombin compared to tryp-
sin and plasmin. We rationalized our results based on
the assumption that all modi®ed P1 side-chains would
still bind into the S1 pocket.
In the urea derivatives 13 to 16 the whole -NH-CO-
NR R -group lies within a plane and probably causes
steric hindrance, especially in the case of 13. Therefore,
the urea derivatives are much less active than com-
pounds 10 to 12.
1
2
The higher anity of compound 17 compared to 9 is quite
unexpected since the less electronegative sulfur in the
thioamide should form a weaker hydrogen bond to the
backbone amide of Gly 219 than the oxygen in the for-
mamide derivative. A possible explanation could be that
the less polar sulfur can better cope with the hydrophobic
environment created by the Boc protecting group. Di€er-
ences in the region around amino acid 217 (Glu in
The P1 side-chains of the compounds described in the
present paper are di€erent: most of them are too large
to ®t into the S1 pocket (e.g., 12 or 13), but even for the
smallest ones (9 or 18), no satisfactory binding mode for
the formamide moiety within the S1 pocket could be
found by molecular modeling studies.

A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2297

2
298
A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
thrombin, Ser in trypsin and Leu in plasmin) could
account for the increase in selectivity of 17 compared to 9.
at room temperature. A precipitate is formed, which is
®ltered o€ and washed with EtOAc and the ®ltrate is
concentrated in vacuo. The resulting oil is puri®ed by ¯ash
chromatography (9:1, hexane:EtOAc). Boc-d-TMSal-
ONp is obtained as white crystals.
Since X-ray structures have only been determined for
compounds 18 and 21, considerations about the SAR
and the binding modes of the other inhibitors remain
speculative. Further experimental structures are needed
to answer these questions.
51.6 g (113 mmol) of Boc-d-TMSal-ONp is dissolved in
THF and an aqueous solution of equimolar amounts of
proline and Et N is added. After 20 h at room temperature,
3
the THF is removed in vacuo and the aqueous residue is
diluted with water and then extracted several times with
EtOAc. The pH of the aqueous layer is adjusted to 3 by
adding 10% citric acid and extracted several times with
EtOAc. The combined organic layers are washed with
brine, dried over Na SO and concentrated in vacuo.
The colourless oil is crystallized from Et O/hexane to
2
give 31.0 g (76%) of Boc-d-TMSal-Pro-OH 2 as a white
crystalline compound: mp: 176 C.
Conclusions
Replacement of the usual basic residues in P1 of thrombin
inhibitors by non-basic moieties could increase the selec-
tivity of the corresponding molecules over trypsin and
plasmin while still maintaining nM thrombin anities.
The structural data so far available
most of these compounds still bind to the S1 pockets of
the serine proteases.
2
4
1
8b,20b,46
indicate that
31.0 g (86.5 mmol) of 2 is dissolved in 350 mL of EtOAc.
After cooling to 0 C, 12 g (103.5 mmol) of N-hydroxy-
ꢀ
succinimide and 18 g (86.5 mmol) of DCC are added.
In this paper, however, we could show for the ®rst time
that the introduction of a butyl formamide as P-1 side-
chain in a tripeptide±boronate (18) can force the throm-
bin/inhibitor complex to adopt a new binding mode in
which the formamide group points out of the S1 pocket
and forms new hydrogen bonds with Gly 219. The
structure±activity relationship of the other compounds
under investigation indicates that these molecules show
the same binding mode as 18.
ꢀ
additional 15 h at room temperature. The mixture is
recooled to 0 C, the dicyclohexylurea is ®ltered o€ and
The mixture is stirred for 3 h at 0 C and then for an
ꢀ
washed several times with EtOAc. The ®ltrate is washed
with aqueous 0.1 M Na CO and then with aqueous 2%
KHSO . After drying over Na SO and concentration in
2
3
4
2
4
vacuo, 38.8g (98%) of Boc-d-TMSal-Pro-ONSu 3 is
obtained as a white foam: FAB MS 456 (MH ); H NMR
+
1
(
CDCl ) d=0.07 (s, 9H), 0.96 (d, J=7.2Hz, 2H), 1.41 (s,
3
9
4
H), 2.00±2.45 (m, 4H), 2.82 (s, 4H), 3.50±3.85 (m, 2H),
.42±4.60 (m, 1H), 4.77±4.90 (m, 1H), 5.10±5.22 (m, 1H).
Experimental
All compounds were characterized by 300 MHz proton
NMR using a Bruker 360 FT-NMR spectrometer. Che-
mical shifts are expressed as ppm down®eld from tetra-
Boc-D-TMSal-Pro-NH-CH[(CH ) N ]B-OPin 7.
2 4 3
1
methylsilane; J values are expressed in Hz. The H NMR
data of the compounds 6±20 are summarized in Table 5,
the signals for the di€erent side-chains and in the case of
compounds 18±20 also the signals for the N-terminal
group can be found after the experimental details for
the compounds. Fast atom bombardment mass spec-
trometry (Xe, 8 keV) on a VG 70-SE mass spectrometer
was used for the characterization of the reported
(+)-Pinanediol-4-bromo-butane-1-boronate. 10.6 g (60.0
mmol) of 4-bromo-1-butene is reacted with 7.30 g
compounds. Melting points were determined on a Bu
35 instrument. For the chromatographic puri®cation,
the ¯ash chromatography technique was applied using
30±400 mesh silica gel.
È
chi
ꢀ
(
60.0 mmol) of catecholborane at 110 C over 18 h. The
5
crude product is added to 10.2 g (60.0 mmol) of (+)-
pinanediol dissolved in 100 mL of THF. After 1 day at
room temperature, the THF is removed in vacuo and
the residue is puri®ed by ¯ash chromatography (95:5,
hexane:EtOAc) to give 15.3 g (81%) of (+)-pinanediol-
2
Boc-D-TMSal-Pro-ONSu 3.
4
-bromo-butane-1-boronate 4 as a colourless oil: MS
+
1
(
CI) 316 (M ); H NMR (CDCl ) d=0.84 (s, 3 H), 0.84
3
(
t, J=7.2 Hz, 2H), 1.08 (d, J=12 Hz, 1H), 1.29 (s, 3H),
1
2
.38 (s, 3H), 1.50±1.65 (m, 2H), 1.80±2.00 (m, 4H),
.00±2.10 (m, 1H), 2.15±2.42 (m, 2H), 3.40 (t,
J=7.2 Hz, 2H), 4.22±4.30 (m, 1H).
2
9.7 g (113 mmol) of Boc-d-TMSal-OH 1 and 19.0 g
Boc-D-TMSal-Pro-NH-CH[(CH ) Br]B-OPin 6. 4.6 mL
4
2
ꢀ
33.5 mL (53.4 mmol) of n-butyllithium (1.6 M in hexane)
(136.3 mmol) of p-nitro-phenol are dissolved in EtOAc.
ꢀ
After cooling to 0 C, 23.4 g (113 mmol) of DCC is added
and the mixture is stirred for 1 h at 0 C and then for 15 h
of CH Cl in 72 mL of THF is cooled to � 100 C and
2
2
ꢀ
ꢀ
is added over 20 min. After 60 min at � 100 C, a cold

A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2299
ꢀ
diol-4-bromo-butane-1-boronate in 28 mL of THF is
(
� 78 C) solution of 15.3 g (48.6 mmol) of (+)-pinane-
10% Pd/C. After 24h at 72psi, the catalyst is removed by
®ltration and the solution is concentrated in vacuo. The
resulting oil is crystallized from Et O/hexane to give 3.10g
ꢀ
24.3 mmol) of anhydrous ZnCl in 28mL of THF is
added. After an additional 1 h at � 100 C, 3.36 g
2
(
(81%) of Boc-d-TMSal-Pro-NH-CH[(CH ) NH ]B-OPin
2
2 4
2
ꢀ
warmed to room temperature and stirred for 4 h. The
ꢀ
(c=1.0in MeOH); FAB MS 621 (MH ); H NMR
20
ꢀ
added. After 15 min at � 100 C, the reaction mixture is
8 as white crystals: mp: 128±129 C; ‰aŠ =� 59.6
d
+
1
reaction mixture is poured into H O and extracted sev-
2
(CDCl ) d=1.20±2.40 (m, 8H), 2.60±2.65 (m, 2H).
3
eral times with Et O. After drying over Na SO and
2
2
4
removal of the solvent in vacuo, 17.4 g (98%) of (+)-
pinanediol-(S)-1-chloro-5-bromo-pentane-1-boronate 5
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)H]B-OPin 9.
2
4
0.22 mL (5.95 mmol) of formic acid and 530 mg
(5.20 mmol) of acetic acid anhydride are stirred at 60 C
+
1
ꢀ
for 2 h. After cooling down to 0 C, 1.86 g (3.00 mmol)
is obtained as a yellow oil: FAB MS 364 (M ); H NMR
CDCl ) d=0.84 (s, 3H), 0.84 (t, J=7.2 Hz, 2H), 1.17 (d,
ꢀ
(
3
J=12 Hz, 1H), 1.27 (s, 3H), 1.42 (s, 3H), 1.47±2.44 (m,
0H), 3.42 (t, J=7.2 Hz, 2H), 4.33±4.40 (m, 1 H).
of 8 in 2 mL of THF is added and the mixture is stirred
for 18 h at room temperature. The reaction mixture is
poured into ice water and extracted several times with
1
A solution of 4.6 mL (22.0 mmol) of LiN(SiMe ) (1 M in
3
Et O. The combined organic layers are washed with
2
2
ꢀ
22.0 mmol) of n-butyllithium (1.6 M in hexane) is added.
THF) in 32 mL of THF is cooled to � 78 C and 13.8 mL
brine, dried over Na CO and the solvent is removed in
2
3
(
The solution is warmed to room temperature and then
vacuo. 1.75 g (90%) of Boc-d-TMSal-Pro-NH-CH
[(CH ) NHC(O)H]B-OPin 9 is obtained as a white
2
4
ꢀ
pinanediol-(S)-1-chloro-5-bromo-pentane-1-boronate in
20
d
ꢀ
cooled down to � 78 C again. 8.00 g (22.0 mmol) of (+)-
foam; ‰aŠ =� 77.8 (c=0.5 in MeOH); FAB MS 649
+
1
ꢀ
6H), 3.03±3.11 (m, 2H), 7.30±7.45 (m, 1H), 7.99 (s, 1H).
(MH ); H NMR (DMSO, 150 C) d=1.30±2.30 (m,
1
1 mL of THF is added and the mixture is stirred for 1 h
ꢀ
at � 78 C, then for 18 h at room temperature.
ꢀ
again. 13mL (5.1N solution, 66.3mmol) of HCl in dioxan
After this period, the reaction mixture is cooled to � 78 C
ꢀ
is added and the solution stirred for 1 h at � 78 C and
then for 2 h at room temperature.
ꢀ
The reaction mixture is cooled down to � 20 C. 10.0 g
(22.0 mmol) of 3 dissolved in 32 mL of CH Cl and 6.1mL
2 2
(
44.0 mmol) of triethylamine are added, and the solution is
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)Me]B-OPin
2
10.
4
ꢀ
perature, the mixture is stirred for an additional 2.5 h, then
stirred at � 20 C for 1 h. After warming up to room tem-
®ltrated and the solvent is removed in vacuo. The residue is
diluted with Et O/H O and extracted several times with
2
2
Et O. After drying over Na SO and concentration in
2
2
4
vacuo, 10.3 g (68%) of Boc-d-TMSal-Pro-NH-CH[(CH )
2
4
+
1
Br]B-OPin 6 is obtained: FAB MS 684 (MH ); H NMR
CDCl ); d=1.25±1.58 (m, 6H), 2.92±3.11 (m, 2H).
(
3
Boc-D-TMSal-Pro-NH-CH[(CH ) N ]B-OPin 7. 5.00 g
2
20 mL (0.25 mmol) of pyridine and 18 mL (0.25 mmol) of
4
3
ꢀ
(7.30 mmol) of 6 is dissolved in 10 mL of DMSO and 960
acetyl chloride in 2 mL of CH Cl are cooled to 0 C,
155.2 mg (0.25 mmol) of 8 in 1 mL of CH Cl is added
2 2
2
2
mg (14.6 mmol) of sodium azide is added. The mixture is
stirred for 7 h at room temperature. EtOAc/ice water is
added and the solution extracted several times with EtOAc.
After drying over Na SO and concentration in vacuo,
and the solution is stirred for 1 h. Water is added and the
product extracted several times with Et O. The com-
2
bined organic layers are washed with brine, dried over
Na SO and concentrated in vacuo. 161 mg (97%) of
2
4
4
.40 g (93%) of Boc-d-TMSal-Pro-NH-CH[(CH ) N ]B-
2
4
3
+
2
4
OPin 7 is obtained as a white foam: FAB MS 647 (MH );
1
Boc-d-TMSal-Pro-NH-CH[(CH ) NH-C(O)Me]B-OPin
2
4
20
d
ꢀ
H NMR (CDCl ) d=1.21±2.48 (m, 6H), 3.78±3.92 (m,
10 is obtained as an oil; ‰aŠ =� 62.6 (c=0.5 in MeOH);
3
+
2H).
MH =663.
Boc-D-TMSal-Pro-NH-CH[(CH ) NH ]B-OPin 8.
2
The following compounds (Boc-d-TMSal-Pro-NH-
CH[(CH ) NHC(O)R]B-OPin) are prepared analogously
from the corresponding acid chlorides or bromides:
4
2
2
4
4.00 g (6.19 mmol) of the azide 7 is dissolved in 30 mL of
EtOAc and hydrogenated in the presence of 0.5 g of

2
300
A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2
1 mL of H
(
48 mg (0.40 mmol) of 16 and 0.4 mL of 1N HCl in
O is warmed to 50 C. After 5 min, 33.2 mg
2
0
MH+
FAB±MS
No.
R
yield
%)
‰aŠ
ꢀ
0.40 mmol) of potassium cyanate is added in small
d
(c=0.5 in
MeOH)
2
(
ꢀ
portions. After 6 h at 50 C water is added and the pro-
duct extracted with EtOAc. The combined organic layers
� 55.8ꢀ
1
1
1
1
2
3
Et
i-Pr
NMe2
95
94
42
677
691
692
ꢀ
are dried over Na
¯ash chromatography (1:9, hexane/EtOAc), 178 mg
67%) of Boc-d-TMSal-Pro-NH-CH-[(CH ) NHC(O)
SO and concentrated in vacuo. After
2 4
� 60.2
� 64.2ꢀ
(
2
4
20
ꢀ
NH ]B-OPin 16 is obtained; ‰ꢀŠ =� 71.7 (c=0.5 in
2
d
MeOH); FAB MS 664 (MH ); H NMR (DMSO,
+
1
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)Me]B-OPin
2
4
1
ꢀ
.78 (s, 3H), 2.72±2.81 (m, 2H), 6.95±7.05 (m, 1H).
ꢀ
4.98 (m, 2 H), 5.50±5.59 (m, 2 H).
1
1
0. H NMR (DMSO, 150 C) d=1.12±2.35 (m, 6H),
120 C) d=1.20±1.58 (m, 6 H), 2.91±2.99 (m, 2 H), 4.87±
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)Et]B-OPin
2
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(S)H]B-OPin 17.
2 4
4
1
ꢀ
11. H NMR (DMSO, 120 C) d=1.01 (t, J=7.2 Hz,
3H), 1.22±1.65 (m, 6H), 2.16 (q, J=7.2 Hz, 2H), 3.00±
3.06 (m, 2H), 7.00±7.27 (m, 1H).
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)i-Pr]B-OPin
4
2
1
ꢀ
H), 1.22±1.64 (m, 6H), 2.21±2.35 (m, 1H), 2.98±3.07
1
6
2. H HMR (DMSO, 120 C) d=1.01 (d, J=7.8 Hz,
(
m, 2H), 6.99±7.07 (m, 1H).
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)NMe ]B-Opin
2
To 227 mg (0.35 mmol) of 9 dissolved in 2.5 mL of toluene
70.8 mg (0.175 mmol) of Lawesson's reagent is added.
After stirring for 1.5 h at room temperature, the solvent
is removed in vacuo. The residue is dissolved in EtOAc,
aluminium oxide is added and the mixture is stirred for
30 min. The aluminium oxide is removed by ®ltration, the
reaction mixture is concentrated in vacuo and the residue
2
4
1
13. H NMR (DMSO) d=1.18±1.60 (m, 6H), 2.84 (s,
6H), 3.11±3.19 (m, 2H), 4.59±4.63 (m, 1H).
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)NHCH ]B-
4
2
3
OPin 14.
crystallized from Et O. 110 mg (47%) of Boc-d-TMSal-
2
Pro-NH-CH[(CH ) NHC(S)H]B-OPin 17 is obtained as
2
4
ꢀ
MeOH); FAB MS 665 (MH ); H NMR (DMSO,
20
d
ꢀ
white crystals; mp: 123±125 C; ‰aŠ =� 74.8 (c=0.5 in
+
1
ꢀ
.87 (m, 2H), 8.50 (s, 1H).
1
4
50 C) d=1.22±2.25 (m, 6H), 3.30±3.40 (m, 2H), 4.78±
1
0
24 mg (0.20 mmol) of 8 in 3 mL of THF is cooled to
C and 12 mL (0.20 mmol) of methyl isocyanate is
ꢀ
added. After stirring 3 h at room temperature, the solvent
Cbz-D-TMSal-Pro-NH-CH[(CH ) NHC(O)H]B-OPin18.
2 4
is removed in vacuo and 128mg (94%) of Boc-d-TMSal-
Pro-NH-CH[(CH ) NH-C(O)NHCH ]B-OPin
14
is
2
4
3
20
ꢀ
MeOH); FAB MS 678 (MH ); H NMR (DMSO,
obtained as a white foam; ‰aŠ =� 62.2 (c=0.5 in
d
+
1
ꢀ
.95±3.01 (m, 2H), 5.18±5.30 (m, 2H).
1
2
20 C) d=1.22±1.59 (m, 6H), 2.57 (d, J=5.4 Hz, 3H),
Analogously, Boc-D-TMSal-Pro-NH-CH[(CH ) NHC
4
2
(
O)NHEt]B-OPin 15. is obtained from ethyl isocyanate
ꢀ
324 mg (0.50 mmol) of 9 dissolved in 3 mL of acetic acid:
concd HCl (9:1) is stirred for 3 h at room temperature.
The solvent is removed in vacuo, and the white foam is
dissolved in 6 mL of THF. After cooling to 0 C, 146 mL
(1.05 mmol) of triethylamine and 82 mL (0.525 mmol) of
benzyl chloroformate are added and the mixture is stir-
2
d
0
in 87% yield; ‰aŠ =� 65.6 (c=0.5 in MeOH); FAB MS
+
1
ꢀ
6
3
2
92 (MH ); H NMR (DMSO, 150 C) d=0.92±1.04 (m,
H), 1.25±1.58 (m, 6H), 2.95±3.07 (m, 4H), 5.22±5.32 (m,
H).
ꢀ
Boc-D-TMSal-Pro-NH-CH[(CH ) NHC(O)NH ]B-OPin
2
red for 3 h. The reaction mixture is poured into H
and extracted several times with Et O. The combined
organic layers are dried over Na SO and concentrated
2
O
2
4
16.
2
2
4
in vacuo. After ¯ash chromatography (92:8, EtOAc:
EtOH), 208 mg (71%) of Cbz-d-TMSal-Pro-NH-CH-
[
(CH ) NHC(O)H]B-OPin 18 is obtained as a white
2 4
20
ꢀ
foam; ‰aŠ =� 55.0 (c=0.46 in MeOH); FAB MS 683
d
+
1
ꢀ
H), 3.00±3.08 (m, 2H), 4.98; 5.06 (2d, J=12 Hz, je 1H),
(
6
MH ); H NMR (DMSO, 150 C) d=1.27±1.54 (m,
7
.28±7.31 (m, 5H), 7.30±7.43 (m, 1H); 7.86 (s, 1H).

A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2301
i-BuC(O)-D-TMSal-Pro-NH-CH[(CH ) NH ]B-OPin 20
2
380 mg (0.77 mmol) of these white crystals are dissolved
in 7.7 mL of EtOH. 480 mL of 2N HCl is added and the
mixture is hydrogenated in the presence of 77 mg of
4
2
1
0% Pd/C. After 2.5 h, the catalyst is removed by ®ltra-
tion and the solution is concentrated in vacuo. The
resulting oil is crystallized from CH Cl /Et O to give
2
2
2
3
15 mg (81%) of i-BuC(O)-d-TMSal-Pro-NH-CH[(CH )
2
4
20
ꢀ
NH ]B(OH)2 21: ‰aŠ =� 60.0 (c=0.51 in MeOH);
2
d
+
1
FAB MS 543 (MH ester with thioglycerine matrix); H
NMR (DMSO) d=0.04 (M, 9H), 0.91 (d, J=7.2 Hz,
6H), 0.95±1.02 (m, 3H), 1.31±1.70 (m, 6H), 1.80±2.12 (m,
6H), 2.52 (m, 1H), 2.65±3.20 (m, 2H), 2.73±2.81 (m, 2 H),
3.76±3.72 (m, 2H), 4.35±4.43 (m, 1H), 4.47±4.60 (m, 1H),
7.30±7.50 (m, 2H), 7.70±8.10 (m, 2H).
.
HCl H-D-TMSal-Pro-NH-CH[(CH ) N ]B-Opin. 5.17 g
2
4
3
(
8.00 mmol) of Boc-d-TMSal-Pro-NH-CH[(CH ) N ]B-
2
4
3
OPin 7 is dissolved in 40 mL of AcOH:concd HCl (9:1)
and stirred at room temperature for 1.5 h. AcOH and
HCl are removed in vacuo and the residue crystallized
.
from Et O. 4.32 g (93%) of HCl H-d-TMSal-Pro-NH-
Remark to the characterization of 21 with FAB MS: the
+
2
CH[(CH ) N ]B-OPin are obtained as white crystals.
MH of the free boronic acids cannot be seen if the
matrix used is thioglycerine. Thioglycerine and boronic
acid react in the mass spectrometer and the character-
2
4
3
i-BuC(O)-D-TMSal-Pro-NH-CH[(CH ) N ]B-OPin 19.
2
.17 g (2.00 mmol) of these crystals are dissolved in 6 mL
of THF and cooled to 0 C. 258 mL (2.10 mmol) iso-
valeryl chloride and 285 mL (4.20 mmol) triethylamine
are added. After stirring for 1 h, the reaction mixture is
poured into ice water and extracted several times with
4
3
+
1
istic peak to be seen is MH +72. This peak was
observed consistently for all the free boronic acids
which were prepared in our labs.
ꢀ
Enzyme inhibition kinetics
Et O. The combined organic layers are dried over
2
Na SO and concentrated in vacuo. Lyophilisation with
4
Human a-thrombin was puri®ed from human plasma
according to published methods, which involved the
2
47
t-BuOH gave 1.21 g (96%) i-BuC(O)-d-TMSal-Pro-
2
0
ꢀ
MeOH); FAB MS 631 (MH ); H NMR (DMSO)
48
NH-CH[(CH ) N ]B-OPin 19: ‰aŠ =� 30.3 (c=0.6 in
puri®cation of prothrombin, the generation of thrombin
from prothrombin by using the venom from Oxyuranus
scutellatus,49 and the puri®cation of thrombin on S-
2
4
3
d
+ 1
d=0.92 (d, J=7.2 Hz, 6H), 1.25±2.14 (m, 9H), 3.25±
50
3.31 (m, 2H).
Sepharose (Pharmacia, Uppsala, Sweden). The isolated
thrombin was free of proteolytically degraded forms, as
judged by SDS-polyacrylamide electrophoresis, and was
found to be >95% active as assessed by active site titration
i-BuC(O)-D-TMSal-Pro-NH-CH[(CH ) NH ]B-OPin 20.
2
4
2
196 mg (0.31 mmol) of the azide 19 is dissolved in
5
1
3
.1 mL of Et O. 186 mL of 2N HCl is added and the
with 4-methylumbelliferyl-p-guanidinobenzoate (Sigma).
2
mixture is hydrogenated in the presence of 62mg of 10%
Pd/C. After 3.5 h, the catalyst is removed by ®ltration and
the solution is concentrated in vacuo. 145 mg (73%) of
i-BuC(O)-d-TMSal-Pro-NH-CH[(CH ) NH ]B-OPin 20
Bovine pancreatic trypsin (Sigma) and human plasmin
(KABI, Moelndal, Sweden) were titrated by the same
method. The substrates Pefachrome TH (2-AcOH-H-d-
CHG-Ala-Arg-pNA), Pefachrome PL (2-AcOH-H-d-Ala-
CHT-Lys-pNA), and Pefachrome TRY (Bz-Gly-Arg-
pNA) were from Pentafarm.
2
4
2
20
ꢀ
is obtained: ‰aŠ =� 74.6 (c=0.24 in MeOH); FAB MS
d
+
1
6
05 (MH ); H NMR (DMSO) d=0.89 (d, J=7.2 Hz,
6H), 1.32±2.10 (m, 9H), 2.71±2.78 (m, 2H), 7.68±7.94
(
m, 2H).
Inhibitors were dissolved in cremophor:ethanol (1:1) or
Me SO and diluted with distilled water to yield a 1 mM
2
i-BuC(O)-D-TMSal-Pro-NH-CH[(CH ) NH ]B(OH) 21.
2
stock solution. Further dilutions were made into the assay
bu€er (100mM sodium phosphate bu€er, pH 7.4, con-
taining 100 mM NaCl and 0.1% bovine serum albumin).
2
4
2
ꢀ
Kinetic assays were performed at 25 C using a microwell
plate; each well contained 50 mL of substrate, 100 mL of
inhibitor, and 100 mL of enzyme in bu€er. Final con-
centration of substrate and enzyme were as follows:
1
60 pM a-thrombin and 100 mM Pefachrome TH
(
K =6.9 mM), 800 pM human plasmin and 200 mM Pefa-
m
8
.1 mL (8.10 mmol) of borotrichloride (1 M solution in
ꢀ
chrome PL (K =66.4 mM), and 260 pM bovine pancrea-
m
CH Cl ) in 8.1 mL of CH Cl is cooled to � 78 C. A
tic trypsin and 500 mM Pefachrome TRY (K =167.7
2
2
2
2
m
ꢀ
6.2 mL of CH Cl is added. The reaction mixture is
stirred 10 min at � 78 C and 1 h at 0 C, then poured into
cold solution (� 78 C) of 1.02 g (1.62 mmol) of 19 in
ꢀ
ice water and extracted several times with Et O. The
mM). K values were determined as previously descri-
m
bed.52 Assays were initiated by adding enzyme to solu-
tions containing inhibitor and substrate. The release of
p-nitroaniline by hydrolysis of the substrate was followed
for 30min by measuring the increase in optical density at
405 nM with a Thermomax microwell kinetic reader
(Molecular Devices, Menlo Park, CA). When the inhibited
steady-state rate was achieved rapidly, the inhibition
1
2
2
ꢀ
2
combined organic layers are washed with brine, dried over
Na CO and concentrated in vacuo. The resulting oil is
crystallized from Et O/hexane to give 520 mg (65%) of the
2
3
2
free boronic acid as white crystals.

2
302
A. von Matt et al. / Bioorg. Med. Chem. 8 (2000) 2291±2303
2. Vu, T.-K. H.; Hung, D. T.; Wheaton, V. I.; Coughlin, S. R.
Cell 1991, 64, 1057.
3. Harker, L. A.; Hanson, S. P.; Kelly, A. B. Thromb. Hae-
most. 1997, 78, 736.
4
5
6
1
3
. Hirsh, J. Fibrinolysis 1995, 9, 66.
. Hirsh, J.; Fuster, V. Circulation 1994, 89, 1469.
. For reviews see: (a) Menear, K. Exp. Opin. Invest. Drugs
999, 8, 1373. (b) Vacca, J. P. Annu. Rep. Med. Chem. 1999,
3, 81. (c) Stone, S. R. Trends Cardiovasc. Med. 1995, 5, 134.
d) Kimball, S. D. Curr. Pharm. Des. 1995, 1, 441. (e) Tappar-
elli, C.; Metternich, R.; Ehrhardt, C.; Cook, N. S. Trends
Pharmacol. Sci. 1993, 14, 366. (f) Das, J.; Kimball, S. D.
Bioorg. Med. Chem. 1995, 3, 999.
Scheme 6.
7
8
. Bush, L. R. Cardiovasc. Drug Rev. 1991, 9, 247.
. Banner, D. W.; Hadvary, P. J. Biol. Chem. 1991, 266,
constant (K ) was determined by ®tting the data by
i
53
20085.
. (a) Preville, P.; He, J. X.; Tarazi, M.; Siddiqui, M. A.;
Cody, W. L.; Doherty, A. M. Bioorg. Med. Chem. Lett. 1997,
, 1563. (b) Teger-Nilsson, A.-C.; Bylund, R.; Gustafsson, D.;
weighted linear regression to the Dixon equation. For
slow, tight-binding inhibitors, the mechanism of inhibition
could be described by Scheme 6, where E, S, P and I are
the enzyme, substrate, product (p-nitroaniline) and
inhibitor, respectively, and kon and ko€ are the association
and dissociation rate constants for the inhibition.
Progress-curve data for the formation of p-nitroaniline
in the presence of di€erent concentrations of inhibitor
were ®tted by non-linear regression to the equation for the
mechanism presented in Scheme 6. These analyses yielded
estimates for the apparent values of k , k and Ki
9
7
Gyzander, E.; Eriksson, U. Thromb. Res. 1997, 85, 133.
10. (a) Eriksson, H.; Eriksson, U. G.; Frison, L.; Thorsen, M.
Thromb. Haemost. 1997, Suppl., 591. (b) Gustafsson, D.;
Antonsson, T.; Bylund, R.; Eriksson, R.; Gyzander, E.; Nils-
son, I.; Elg, M.; Mattsson, C.; Deinum, J.; Pehrsson, S.;
Karlsson, O.; Nilsson, A., Sorensen, H. Thromb. Haemost.
1
998, 79, 110.
1. Hilpert, K.; Ackermann, J.; Banner, D. W.; Gast, A.;
1
Gubernator, K.; Hadvary, P.; Labler, L.; Mu
on
o€
È
ller, K.; Schmid,
G.; Tschopp, T. B.; van de Waterbeemd, H. J. Med. Chem.
which were corrected for the presence of substrate as
54
described by Morrison and Walsh to give the true
values. The kinetic constants were determined in triplicate
and expressed as meanÆS.D.
1
1
994, 37, 3889.
2. Iwanowicz, E. J.; Lau, W. F.; Lin, J.; Roberts, D. G. M.;
Seiler, S. M. J. Med. Chem. 1994, 37, 2122.
3. Obst, U.; Gramlich, V.; Diederich, F.; Weber, L.; Banner,
1
D. W. Angew. Chem. 1995, 107, 1874; Angew. Chem., Int. Ed.
Engl. 1995, 34, 1739.
Crystallography
1
4. (a) Tucker, T. J.; Lumma, W. C.; Lewis, S. D.; Gardell, S.
Protein puri®cation and crystallization. Human a-
thrombin was prepared for crystallization according to
the literature. Crystals were grown using the hanging
drop method following a procedure similar to the one
described in the literature.56 The crystals were grown in
a 10-fold excess of the inhibitors 18 and 21, respectively,
J.; Lucas, B. J.; Baskin, E. P.; Woltmann, R.; Lynch, J. J.;
Lyle, L. A.; Appleby, S. D.; Chen, I.-W.; Dancheck, K. B.;
Vacca, J. P. J. Med. Chem. 1997, 40, 1565. (b) Feng, D.-M.;
Gardell, S. J.; Lewis, S. D.; Bock, M. G.; Chen, Z.; Freidinger,
R. M.; Naylor-Olsen, A. M.; Ramjit, H. G.; Woltmann, R.;
Baskin, E. P.; Lynch, J. J.; Lucas, R.; Shafer, J. A.; Dancheck,
K. B.; Chen, I.-W.; Mao, S.-S.; Krueger, J. A.; Hare, T. R.;
Mulichak, A. M.; Vacca, J. P. J. Med. Chem. 1997, 40, 3726.
5
5
and after micro-seeding they grew to a ®nal size of
3
0.4Â0.3Â0.3 mm .
(
c) Tucker, T. J.; Lumma, W. C.; Lewis, S. D.; Gardell, S. J.;
Data processing and re®nement. The X-ray intensity data
were collected on a FAST television area detector with
an Enraf-Nonius FR571 rotating anode operating at
Lucas, B. J.; Sisko, J. T.; Lynch, J. J.; Lyle, L. A.; Baskin, E.
P.; Woltmann, R.; Appleby, S. D.; Chen, I.-W.; Dancheck, K.
B.; Naylor-Olsen, A. M.; Krueger, J. A.; Cooper, C. M.;
Vacca, J. P. J. Med. Chem. 1997, 40, 3687. (d) Brady, S. F.;
Stau€er, K. J.; Lumma, W. C.; Smith, G. M.; Ramjit, H. G.;
Lewis, S. D.; Lucas, B. J.; Gardell, S. J.; Lyle, E. A.; Appleby,
S. D.; Cook, J. J.; Holahan, M. A.; Stranieri, M. T.; Lynch, J.
J.; Lin, J. H.; Chen, I.-W.; Vastag, K.; Naylor-Olsen, A. M.;
Vacca, J. P. J. Med. Chem. 1998, 41, 401.
15. Sall, D. J.; Bastian, J. A.; Briggs, S. L.; Buben, J. A.;
Chirgadze, N. Y.; Clawson, D. K.; Denney, M. L.; Giera, D.
D.; Gi€ord-Moore, D. S.; Harper, R. W.; Hauser, K. L.;
Klimkowski, V. J.; Kohn, T. J.; Lin, H.-S.; McCowan, J. R.;
Palkowitz, A. D.; Smith, G. F.; Takeuchi, K.; Thrasher, K. J.;
Tinsley, J. M.; Utterback, B. G.; Yan, S.-C. B.; Zhang, M. J.
Med. Chem. 1997, 40, 3489.
4
0kV and 80mA. Data were measured at room tempera-
ꢀ
out-angle of 20 , and a crystal-to-detector distance of
ture in 100-s frames of width 0.15 in o, a detector-swing-
ꢀ
5 mm. The evaluation of the measured intensities was
5
performed by the program MADNES.
5
7
Structure
solution, re®nement and analysis were performed with
5
8
59
the programs O and CNS.
Acknowledgements
We would like to thank T. Thai, H. Kaiser, W. Lu
Gfeller and B. Ga€ner for their skilful technical assistance.
È
di, P.
1
6. (a) Sanderson, P. E. J.; Cutrona, K. J.; Dorsey, D. B.;
Dyer, D. L.; McDonough, C. M.; Naylor-Olsen, A. M.; Chen,
I.-W.; Chen, Z.; Cook, J. J.; Gardell, S. J.; Krueger, J. A.;
Lewis, S. D.; Lin, J. H.; Lucas, B. J.; Lyle, E. A.; Lynch, J. J.;
Stranieri, M. T.; Vastag, K.; Shafer, J. A.; Vacca, J. P. Bioorg.
Med. Chem. Lett. 1998, 817. (b) Sanderson, P. E.; Lyle, T. A.;
Cutrona, K. J.; Dyer, D. L.; Dorsey, B. D.; McDonough, C.
M.; Naylor-Olsen, A. M.; Chen, I.-W.; Chen, Z.; Cook, J. J.;
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