Structure-based design of novel potent nonpeptide thrombin inhibitors

Article abstract of DOI:10.1021/jm0109513

The clinical syndromes of thromboembolism are evoked by an excessive stimulation of the coagulation cascade. In this context, the serine protease thrombin plays a key role. Considerable efforts have therefore been devoted to the discovery of safe, orally active inhibitors of this enzyme. On the basis of the X-ray crystal structure of the peptidelike thrombin inhibitor NAPAP complexed with bovine thrombin, we have designed a new structural class of nonpeptidic inhibitors employing a 1,2,5-trisubstituted benzimidazole as the central scaffold. Supported by a series of X-ray structure analyses, we optimized the activity of these compounds. Thrombin inhibition in the lower nanomolar range could be achieved although the binding energy mainly results from nonpolar, hydrophobic interactions. To improve in vivo potency, we increased the overall hydrophilicity of the molecules by introducing carboxylate groups. The very polar compound 24 (BIBR 953) exhibited the most favorable activity profile in vivo. This zwitterionic molecule was converted into the double-prodrug 31 (BIBR 1048), which showed strong oral activity in different animal species. On the basis of these results, 31 was chosen for clinical development.

Full text of DOI:10.1021/jm0109513

J . Med. Chem. 2002, 45, 1757-1766
1757
Str u ctu r e-Ba sed Design of Novel P oten t Non p ep tid e Th r om bin In h ibitor s
Norbert H. Hauel,* Herbert Nar, Henning Priepke, Uwe Ries, J ean-Marie Stassen, and Wolfgang Wienen
Boehringer Ingelheim Pharma KG, Research Division, Birkendorfer Strasse 65, D-88397 Biberach/ RISS, Germany
Received J une 11, 2001
The clinical syndromes of thromboembolism are evoked by an excessive stimulation of the
coagulation cascade. In this context, the serine protease thrombin plays a key role. Considerable
efforts have therefore been devoted to the discovery of safe, orally active inhibitors of this
enzyme. On the basis of the X-ray crystal structure of the peptidelike thrombin inhibitor NAPAP
complexed with bovine thrombin, we have designed a new structural class of nonpeptidic
inhibitors employing a 1,2,5-trisubstituted benzimidazole as the central scaffold. Supported
by a series of X-ray structure analyses, we optimized the activity of these compounds. Thrombin
inhibition in the lower nanomolar range could be achieved although the binding energy mainly
results from nonpolar, hydrophobic interactions. To improve in vivo potency, we increased the
overall hydrophilicity of the molecules by introducing carboxylate groups. The very polar
compound 24 (BIBR 953) exhibited the most favorable activity profile in vivo. This zwitterionic
molecule was converted into the double-prodrug 31 (BIBR 1048), which showed strong oral
activity in different animal species. On the basis of these results, 31 was chosen for clinical
development.
In tr od u ction
In this field, considerable progress has been made
during the past decade.4 The early work focused on
structures based on the classical tripeptide motif D-Phe-
Pro-Arg,5 whereas during recent years nonpeptide in-
hibitors were designed as a strategy to improve phar-
macokinetic properties of the drug molecules.6 In this
context, compounds were synthesized that incorporate
a bicyclic lactam,7 a pyridone,8 or a pyrazinone ring9 as
a P2 surrogate within the tripeptide scaffold. Others
have reported inhibitors with a piperidine,10 a 1,4-
disubstituted phenylene,11 a 1,3-disubstituted phen-
ylene,12 or a 1,3-disubstituted toluene ring13 as the core
structure.
Thromboembolic disorders are the major cause of
mortality and morbidity in Western societies. Closely
linked to the clinical syndromes of thromboembolism is
an excessive stimulation of the coagulation cascade.1
This activation involves two crucial steps that together
are referred to as the common coagulation pathway: (i)
the generation of thrombin via the prothrombinase
complex, consisting of factor Va, Xa, and phospholipids
and (ii) the proteolytic cleavage of fibrinogen by throm-
bin, giving rise to the insoluble clot matrix, which
consists of fibrin. Thrombin in addition induces platelet
activation and also triggers a wide range of effects
secondary to thrombosis, e.g., vascular smooth muscle
cell and fibroblast proliferation, monocyte chemotaxis,
and neutrophil adhesion.
Current anticoagulant therapy involves heparin or
low molecular weight heparins and, for oral treatment,
coumadin.2 However, the clinical use of these agents has
several important drawbacks. Heparins must be ad-
ministered parenterally, and the use of coumadin needs
frequent monitoring. On the basis of its biological mode
of action, it displays a slow onset of action and its
efficacy is dependent on the patient’s nutritional status.
Recently, the direct thrombin inhibitor hirudin was
tested for the prevention of deep venous thrombosis
after major orthopedic surgery, where superiority over
heparin and low molecular weight heparin could be
demonstrated.3 However, because of its peptidic nature,
this compound must be given parenterally, providing a
major obstacle for the subacute and chronic treatment
of thromboembolic diseases. Therefore, research efforts
were aimed at the identification of synthetic, orally
active, direct thrombin inhibitors.
The starting point of our search for noncovalent,
nonpeptide thrombin inhibitors was the X-ray crystal
structure of the bovine thrombin complex formed with
the peptidelike, benzamidine-based inhibitor NAPAP14
(1), which shows the conformation of the enzyme-bound
inhibitor and its interaction with residues of the active
site cleft. The amidine group forms a bidentate salt
bridge with the carboxylate of Asp189. Hydrophobic
interactions are formed by the piperidine ring and the
naphthyl moiety with the proximal (P) and the distal
(D) pocket of the thrombin active site, respectively.
Additionally, the bridging glycine moiety of NAPAP, in
analogy to peptidic serine protease substrates, forms the
canonical hydrogen-bonding pattern with residues Trp215
and Gly216 at the rim of the specificity (S1) pocket.
On the basis of this information, we designed a
number of cyclic scaffolds as surrogates for the central
glycine in the NAPAP molecule and, as we did so,
neglected the hydrogen bonds that are formed by the
glycine amino and carbonyl groups with the enzyme.
One of the most interesting scaffolds we found was the
trisubstituted benzimidazole as exemplified in com-
pound 2 (Chart 1).
* To whom correspondence should be addressed. Tel.:
+49/7351/54-4306. Fax: +49/7351/54-5181. E-mail: norbert.hauel@
bc.boehringer-ingelheim.com.
We investigated the exact binding mode of this
inhibitor by determining a crystal structure of a complex
10.1021/jm0109513 CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/26/2002

1758 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Hauel et al.
Ch a r t 1
Ch a r t 2
analyses of inhibitor-thrombin complexes. We synthe-
sized a series of analogues of compound 2 according to
the general formula shown in Chart 2. Refinement of
this compound in order to increase its inhibitory po-
tency, its tolerability, and its pharmacokinetic proper-
ties was the goal of this study. In this paper, we describe
our progress toward this goal.
with human R-thrombin. As can be seen from Figure 1,
the benzimidazole appears to be a most favorable
template to place the three essential substituents into
the right positions.
Compound 2 interacts via a salt bridge with Asp189
in the same way as NAPAP. The terminal phenyl ring
is bound by a hydrophobic interaction in the D-pocket,
and the N-methyl group nicely fits into the P-pocket.
As was expected, there is no additional hydrogen bond
between the inhibitor and the enzyme, which in part
may explain why in our test system compound 2 was
about 1 order of magnitude less active (IC50 ) 1.5 µM)
than NAPAP (IC50 ) 0.2 µM).
When given intravenously to anaesthetized rats,
compound 2 showed a considerably longer half-life than
NAPAP (data not shown); it was, however, not well-
tolerated. Even the dose of 1 mg/kg i.v. produced strong
cardiovascular side effects in terms of reduced blood
pressure and heart rate.15
Despite these shortcomings, compound 2 served as an
important lead structure for us. Our further optimiza-
tion efforts were supported by a number of X-ray
Syn th etic Ch em istr y
The sulfonamides 2-6 of Table 1 were synthesized
as shown in Scheme 1. The benzimidazole intermediates
25a -d were obtained by acylation of the respective
phenylenediamine starting materials with 4-cyanophen-
ylacetic acid followed by ring closure in boiling concen-
trated acetic acid. Reduction of the nitro groups and
subsequent sulfonation with benzenesulfonyl chloride
gave intermediates 26a -d , which were converted into
the benzamidines 2-5 by the Pinner reaction. Com-
pound 6 was prepared by methylation of the sulfonyl-
amide nitrogen of 26b prior to the Pinner reaction.
Compounds 7-13 were synthesized in an analogous
procedure by first sulfonating intermediate 25b with the
respective arylsulfonyl chlorides and second converting
the nitriles into the benzamidines (Scheme 2). N-
Alkylation of 27 with ethyl bromoacetate and ethyl
4-bromobutyrate followed by Pinner reactions and fi-
nally by hydrolyses of the ester groups afforded the
zwitterionic thrombin inhibitors 14 and 15.
F igu r e 1. Stereo representation of the X-ray analysis of compound 2 (green carbon atoms) in complex with thrombin (blue
carbon atoms). The interactions of the ligand with the protein are mainly hydrophobic in character with the N-methyl group
occupying the P-pocket (residues Trp 60D, His 57, Tyr 60B, and Leu 99) and the phenyl sulfone occupying the D-pocket above
residue Trp 215. The benzamidine moiety binds to Asp 189 in the specificity pocket via a bidentate salt bridge interaction. The
bridging benzimidazole scaffold does not form any polar interactions with the protein.

Design of Novel Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1759
Ta ble 1. Structural Variations of Lead Compound 2
compd
L
R1
R2
Ar
IC50 (µM)a
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
-CH2-
-CH3
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-H
-H
-CH2-COOH
-(CH2)3-COOH
-CH2-COOH
-CH2-COOH
-CH2-COOH
phenyl
phenyl
phenyl
phenyl
phenyl
3-pyridyl
2,5-dimethoxyphenyl
3,5-bis-trifluoromethyl-phenyl
1-naphthyl
2-naphthyl
5-isoquinolyl
8-quinolyl
8-quinolyl
8-quinolyl
1.5
19
2.4
4.0
1.3
-CH2-
-CH2-
-ethyl
-n-propyl
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH2-
-CH2-
-CH2-
1.6
1.8
>100
0.6
-CH2-
-CH2-
-CH2-
-CH2-
24
-CH2-
0.51
0.26
0.12
0.13
0.032
0.058
0.011
-CH2-
-CH2-
-CH2-
-CH2-CH2-
-CH2-O-
-CH2-NH-
8-quinolyl
8-quinolyl
8-quinolyl
a
IC50 for inhibition of human thrombin. For details, see Experimental Section.
Sch em e 1a
Sch em e 2a
a
(a) Arylsulfonyl chloride, pyridine, room temperature; (b) HCl
(gas), ethanol, 0 °C, (NH4)2CO3/ethanol, room temperature; (c)
ethyl bromo-acetate, K2CO3 , DMSO, room temperature; (d) NaOH,
H2O/ethanol, room temperature; (e) ethyl 4-bromo-butyrate, K2CO3,
DMSO, room temperature.
a
(a) (4-Cyano-phenyl)acetic acid, CDI, THF, 50 °C; (b) acetic
acid, reflux; (c) H2, 10% Pd/C, methanol/CH2Cl2; (d) benzenesulfo-
nyl chloride, pyridine, room temperature; (e) HCl (gas), ethanol,
0 °C, (NH4)2CO3/ethanol, room temperature; (f) iodomethane,
K2CO3, acetone, room temperature.
noxyacetic acid, and 4-cyanophenylglycine and subse-
quent ring closure gave the benzimidazole intermediates
28a -c, which were converted in five steps into the
zwitterionic molecules 16-18 in the same way as
described for compound 14.
The same kind of chemistry was applied for the
preparation of sulfonamides 16-18 as illustrated in
Scheme 3. Acylation of 3-amino-4-methylamino-nitroben-
zene with 4-cyanophenylpropionic acid, 4-cyanophe-

1760 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Hauel et al.
Sch em e 3a
Sch em e 4a
a
(a) CDI, N1-methyl-4-nitro-benzene-1,2-diamine, THF, 50 °C;
(b) acetic acid, reflux; (c) H2, 10% Pd/C, methanol/CH2Cl2, room
temperature; (d) quinoline-8-sulfonyl chloride, pyridine, room
temperature; (e) ethyl bromo-acetate, K2CO3, acetone, reflux; (f)
HCl (gas), ethanol, 0 °C, (NH4)2CO3/ethanol, room temperature;
(g) NaOH, H2O/ethanol, room temperature.
a
(a) SOCl2/DMF, reflux; (b) arylamino-alkanoic acid ethyl ester,
THF, triethylamine, room temperature; (c) H2, 10% Pd/C, methanol/
CH2Cl2, room temperature; (d) 3-(4-cyanophenyl)propionic acid or
4-cyanophenoxy-acetic acid or 4-cyanophenyl-glycine, CDI, THF,
50 °C; (e) acetic acid, reflux; (f) HCl (gas), ethanol, 0 °C, (NH4)2CO3/
ethanol, room temperature; (g) NaOH, H2O/ethanol, room tem-
perature.
Ta ble 2. Carboxamide Analogues
Sch em e 5a
compd
A
n
X
IC50 (µM)a
19
20
21
22
23
24
-CH2-
-O-
-NH-
-NH-
-NH-
-NH-
1
1
1
2
3
2
CH
CH
CH
CH
CH
N
0.054
0.33
0.010
0.0054
0.010
0.0093
a
IC50 for inhibition of human thrombin. For details, see
Experimental Section.
Table 2 summarizes those enzyme inhibitors that
employ a carboxamide group (instead of a sulfonamide
group) as a linker between the central template and the
terminal aryl moiety. The synthetic pathway is depicted
in Scheme 4. 4-Methylamino-3-nitrobenzoic acid was
condensed with a variety of arylamino derivatives to
yield intermediates 29a -d . After the nitro groups were
reduced, the resulting phenylene diamines were con-
verted into benzimidazoles in two reaction steps as
already described. Pinner reaction and ester hydrolysis
finally gave the test compounds 19-23.
Scheme 5 illustrates the synthetic route for inhibitor
24, a pyrido analogue of 22. The nitro group of inter-
mediate 29d was reduced, the resulting phenylenedi-
amine derivative was acylated with 4-cyanophenylgly-
cine, ring closure was performed in hot concentrated
acetic acid, and the nitrile was transformed into the
benzamidine 30. This intermediate was converted into
compound 24 by alkaline hydrolysis. Reaction of 30 with
n-hexyl chloroformate in acetone/water yielded the O-n-
hexylcarbamate derivative 31.
a
(a) NaOH, H2O/ethanol, room temperature; (b) n-hexyl chlo-
roformate, K2CO3, THF/H2O, room temperature.
Discu ssion
Starting from lead structure 2, we synthesized ana-
logues 3-5 (Table 1) with substituents R1 of different
lipophilicity at the benzimidazole N-1, to find out their
relative contribution to the binding energy and to
identify the optimum alkyl chain length. Compound 3

Design of Novel Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1761
(R1 ) H) turned out to be 1 order of magnitude less
active than 2, which clearly demonstrates the impor-
tance of the lipophilic/hydrophobic interaction of the
methyl group of 2 with the lipophilic P-pocket formed
by the residues His57, Tyr60A, Leu99, and Trp60D of
thrombin. This finding was not a surprise because from
the X-ray structure (Figure 1) we already had informa-
tion about the localization of the methyl group in the
enzyme active site. We did not, however, expect that
an increase in alkyl chain length (compounds 4 and 5)
would result in a decrease in activity. We had hoped,
instead, that a larger buried hydrophobic surface area
and therefore a stronger van der Waals interaction
would result in stronger ligand binding. The observed
decrease in affinity, albeit very small, may best be
explained by a decrease of entropy due to a loss of
rotational degrees of freedom during formation of the
enzyme-inhibitor complex.16
F igu r e 2. X-ray crystal structure of compound 24 in complex
with thrombin in a surface representation. The lipophilic
potential is mapped on the protein surface. The ligand
interacts in a similar fashion as compound 2 with thrombin
residues of the specificity pocket and the P-pocket. The
D-pocket is occupied by the pyridine ring, while the propionic
acid substituent on the amide nitrogen projects into bulk
solvent without forming further interactions with the protein.
The X-ray structure (Figure 1) clearly shows that the
sulfonamide hydrogen of compound 2 does not take part
in a hydrogen bond with the protein. The N-H bond is
directed out of the binding site into the solvent phase;
N-alkylation should therefore have no influence on
affinity, as was proven with the N-methyl derivative 6.
The inhibitor interaction with the thrombin D-pocket,
formed by residues Leu99, Ile174, and Trp215, was
explored by variations of the terminal aryl substituent
Ar. A selection of some derivatives is listed in Table 1
(compounds 7-13). Substitution of the phenyl group by
3-pyridyl (compound 7) or 2,5-dimethoxyphenyl (com-
pound 8) did not influence affinity, while the 3,5-bis-
trifluoromethyl-phenyl derivative (compound 9) did not
show any activity at a concentration of 100 µM, most
probably due to steric hindrance caused by the two
trifluoromethyl groups. A small increase in affinity as
compared to compound 2 was achieved with the 1-naph-
thyl residue (compound 10), whereas activity dropped
40-fold with the 2-naphthyl group (compound 11),
probably caused by a repulsive interaction with Asn98
at the back of the D-pocket. The 5-isoquinolyl and
8-quinolyl groups (compounds 12 and 13) were as
effective as the 1-naphthyl.
template and the benzamidine moiety (Chart 2) by an
additional atom should generate molecules that fit
equally well into the active site. This was proven by
compounds 16-18 (Table 1). Much to our surprise,
compound 18 even turned out to be 1 order of magnitude
more active than 14. On the basis of X-ray structures,
our hypothesis at this point was that the diatomic linker
facilitates a better positioning of the benzamidine group
in the S1 pocket.
In the molecules of Table 1, the position of the
terminal aryl residue Ar in relation to the central
benzimidazole scaffold is determined by the torsion
angles of the sulfonamide spacer between them. Having
obtained detailed information about enzyme-bound
conformations of a number of inhibitors, we reasoned
that by utilizing a carboxamide instead of the sulfona-
mide spacer the aryl group should also be placed in a
favorable position for strong D-pocket interaction. This
hypothesis was verified by the carboxamide analogues
listed in Table 2. From the three variations of the linker
-CH2-A- (compounds 19-21), again, the amino de-
rivative 21 was the most active, as was the case in the
sulfonamide series in Table 1. Compounds 21-23
demonstrate that the chain length (up to three meth-
ylene groups) between the carboxylate and the amide
nitrogen has only a minor influence on the in vitro
activity. Finally, 24 shows that the terminal phenyl can
be substituted by the more hydrophilic 2-pyridyl group
without substantial loss of activity.
As was reported by others with a different class of
thrombin inhibitors,17 the lipophilicity of the molecules
influences their inhibitory activity when measured in
the presence of blood plasma: the more lipophilic the
inhibitor is, the more pronounced the reduction in
activity will be. The reason for this phenomenon most
probably is plasma protein binding, which reduces the
free inhibitor concentration. We therefore planned to
introduce polar groups into the molecules in order to
increase their hydrophilicity and thereby reduce protein
binding and optimize in vivo activity. To this end, we
synthesized the zwitterionic compounds 14 and 15 by
adding acetic acid and butyric acid, respectively, to the
sulfonamide nitrogen. As inferred from the crystal
structure of the thrombin-2 complex, the carboxylate
groups are located outside the active site in the solvent
phase and do not interfere with inhibitor binding. The
inhibitory activity of both compounds was not reduced
in the presence of plasma proteins (data not shown).
The X-ray structure of 24 in complex with thrombin
is shown in Figure 2. It demonstrates that this inhibitor
and our starting point, compound 2, share a similar
binding mode: The amidine group interacts with Asp189
via a bidentate salt bridge, the central template is bound
to thrombin by a hydrophobic interaction with the
P-pocket, and the pyridine ring is positioned between
Leu99 and Ile174 in the D-pocket. As predicted, there
is no interaction between the enzyme and the carboxy-
late group that is directed out of the binding site into
From X-ray structure analyses of this class of inhibi-
tors in complex with thrombin, we reasoned that
elongation of the linker L between the benzimidazole

1762 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Ta ble 3. Activity Profile of BIBR 953 (24)
Hauel et al.
structural class of nonpeptidic inhibitors employing a
benzimidazole as the central scaffold. Supported by a
series of X-ray structure analyses, we optimized the
inhibitory activity of these compounds in a number of
iterative steps. During this process, enzyme inhibition
in the lower nanomolar range could be achieved al-
though, apart from the salt bridge of the amidinium
group with Asp189 of thrombin, the binding energy
solely results from hydrophobic interactions. To reduce
plasma protein binding, we increased the hydrophilicity
of the inhibitors by introducing a carboxylate group at
a position of the molecules, where it does not interfere
with the thrombin binding site interaction. From this
series of inhibitors, 24 (BIBR 953) exhibited the most
favorable in vivo activity profile following i.v. adminis-
tration to rats. Because of its highly polar, zwitterionic
nature, oral absorption of 24 was insufficient. Therefore,
a number of prodrugs were synthesized, from which 31
(BIBR 1048) exhibited strong and long-lasting antico-
agulant effects after oral administration in different
animal species. On the basis of these encouraging
results, 31 was chosen for development and is currently
undergoing phase II clinical trials in patients with
thromboembolic disorders.
human enzymes
Ki (nM)
human enzymes
Ki (nM)
thrombin
factor Xa
trypsin
4.5 ( 0.2 plasmin
3760 ( 20 tPA
1695 ( 50
45360 ( 10
50.3 ( 0.3 activated protein C 20930 ( 10
a
PTT (human plasma): ED2 ) 0.23 ( 0.021 µM.
F igu r e 3. Dose- and time-dependent effects on aPTT after
oral administration of compound 31 (BIBR 1048) to rhesus
monkeys. Data represent mean ( SEM.
bulk solvent. It is one of the most remarkable charac-
teristics of this inhibitor that it does not make use of
the canonical hydrogen-bonding pattern with the en-
zyme, which is a common feature of almost all high
affinity thrombin inhibitors reported so far. Apart from
the salt bridge with Asp189, the binding energy of 24
solely results from hydrophobic interactions, which
underscores the general importance of this binding force
in drug receptor interactions.18
Because of its strong in vitro activity and its favorable
selectivity profile vs related serine proteases (Table 3),
24 was investigated biologically in depth and turned out
to be a very potent anticoagulant in vivo. Among all of
the inhibitors of this structural class, it exhibited the
strongest activity and the longest duration of action in
anaesthetized rats after i.v. administration. Unlike
compound 2, it was well-tolerated in these animals up
to the highest given dose of 10 mg/kg. It was, however,
not orally active, which is not surprising considering
that it is a very polar, permanently charged molecule
with a logP of -2.4 (n-octanol/buffer, pH 7.4).
In principle, compounds with physicochemical char-
acteristics such as this can be converted into orally
active prodrugs by turning the carboxylate into an ester
group and by masking the amidinium moiety as a
carbamate-ester,19 provided that both the carboxylate
and the amidinium will be restored in vivo by hydrolytic
cleavage of the prodrug. Accordingly, we synthesized a
series of double-prodrugs of this type as exemplified by
31 (Scheme 5), which is a rather lipophilic compound
with a logP value of 2.6 (n-octanol/buffer, pH 7.4). Given
orally to rhesus monkeys, this prodrug 31 exhibited
strong and long-lasting anticoagulant effects as mea-
sured by the activated partial thromboplastin time
(aPTT) ex vivo (Figure 3). A more detailed description
of the biological effects of 24 (BIBR 953) and its prodrug
31 (BIBR 1048) will be published soon.
Exp er im en ta l Section
Ch em istr y. Procedures for the preparation of all final
products are presented below along with representative pro-
cedures for all methods used in the preparation of intermedi-
ates. All solvents and reagents were used without purification
as acquired from commercial sources.
1H nuclear magnetic resonance (NMR) spectra were re-
corded at 200 MHz on a Bruker AC200 spectrophotometer in
the solvent indicated. Chemical shifts are reported in parts
per million relative to tetramethysilane. Melting points were
recorded on a HWS SG 2000 melting point apparatus and are
uncorrected. Elemental analyses were performed on a Heraeus
CHN Rapid elemental analyzer and are within 0.4% of the
calculated values. Mass spectra were recorded on the following
instruments, using the stated ionization methods: Finnigan
MAT 8230 mass spectrometer, electron impact ionization
(YEF); Finnigan TSQ 700 mass spectrometer, electrospray
ionization (cation scan: EKA).
4-(5-Am in o-1-m et h yl-1H -b en zoim id a zol-2-ylm et h yl)-
ben zon itr ile (25b). A solution of (4-cyano-phenyl)acetic acid
(10.1 g, 62.7 mmol) and CDI (10.1 g, 62.3 mmol) in 100 mL of
tetrahydrofuran (THF) was stirred at 50 °C for 30 min. Then,
N1-methyl-4-nitro-benzene-1,2-diamine (10.1 g, 60.4 mmol) in
400 mL of THF was added and the resulting solution was
stirred for another 2 h at 50 °C. About 350 mL of the solvent
was evaporated, water (250 mL) was added, and the precipi-
tate was collected and washed with water and subsequently
with ether to afford 2-(4-cyano-phenyl)-N-(2-methylamino-5-
nitro-phenyl)acetamide (14.8 g, 79%). This intermediate was
dissolved in glacial acid (150 mL) and refluxed for 1 h. The
solution was then concentrated to dryness, and the residue
was vigorously stirred in ammonia solution (1 N, 200 mL),
which gave, after it was filtered, 4-(1-methyl-5-nitro-1H-
benzoimidazol-2-ylmethyl)benzonitrile (13.0 g, 93%). The nitro
group was then reduced by hydrogenation (Pd on charcoal,
10%) in a mixture of methanol and dichloromethane (200 mL
each), to afford the desired 25b (11.6 g, 100%). Low-resolution
mass spectromety (LRMS) (YEF): M+ ) 262. 1H NMR (DMSO-
d6): δ 3.60 (s, 3H), 4.31 (s, 2H), 4.86 (bs, 2H), 6.56 (dd, 1H),
6.71 (d, 1H), 7.12 (d, 1H), 7.47 (d, 2H), 7.79 (d, 2H).
N-[2-(4-Cya n o-b en zyl)-1-m et h yl-1H -b en zoim id a zol-5-
yl]ben zen esu lfon a m id e (26b). A solution of 25b (1.60 g, 6.1
mmol) and benzenesulfonyl chloride (1.10 g, 6.2 mmol) in 30
mL of pyridine was kept at room temperature for 1 h. The
solvent was then evaporated, and the residue was vigorously
Con clu sion
On the basis of the X-ray crystal structure of NAPAP
complexed with bovine thrombin, we designed a new

Design of Novel Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1763
stirred in a mixture of water (20 mL) and ethyl acetate (2 mL).
The solid was isolated by filtration and washed with water
and ether to afford 2.21 g (91%) of the benzenesulfonamide
26b. LRMS (YEF): M+ ) 402. 1H NMR (DMSO-d6): δ 3.68 (s,
3H), 4.37 (s, 2H), 6.95 (dd, 1H), 7.22 (d, 1H), 7.38 (d, 1H), 7.44-
7.73 (m, 5H), 7.49 (d, 2H), 7.78 (d, 2H), 10.09 (s, 1H).
4-[5-(2,5-Dim eth oxy-ben zen esu lfon yla m in o)-1-m eth yl-
1H-ben zoim id a zol-2-ylm eth yl]ben za m id in e Hyd r och lo-
r id e (8). Starting from 25b and 2,5-dimethoxy-benzenesulfo-
nyl chloride, the title compound was synthesized following the
procedures described above. LRMS (EKA): (M + H)+ ) 480.
1H NMR (DMSO-d6): δ 3.66 (s, 6H), 3.88 (s, 3H), 4.37 (s, 2H),
7.00 (dd, 1H), 7.05-7.16 (m, 3H), 7.22 (d, 1H), 7.32 (d, 1H),
7.50 (d, 2H), 7.78 (d, 2H), 9.18 (s, 2H), 9.35 (s, 2H), 9.74 (s,
1H). Anal. (C24H25N5O4S‚HCl) C, H, N.
4-[5-(3,5-Bis-tr iflu or om eth yl-ben zen esu lfon yla m in o)-
1-m eth yl-1H-ben zoim idazol-2-ylm eth yl]ben zam idin e Hy-
dr och lor ide (9). Starting from 25b and 3,5-bis-trifluoromethyl-
benzenesulfonyl chloride, the title compound was synthesized
following the procedures described above. LRMS (EKA): (M
+ H)+ ) 556. 1H NMR (DMSO-d6): δ 3.69 (s, 3H), 4.39 (s, 2H),
6.95 (dd, 1H), 7.26 (d, 1H), 7.40 (d, 1H), 7.48 (d, 2H), 7.78 (d,
2H), 8.23 (s, 2H), 8.47 (s, 1H), 9.15 (s, 2H), 9.34 (s, 2H), 10.04
(s, 1H). Anal. (C24H19F6N5O2S‚HCl) C, H, N, S.
4-[1-Meth yl-5-(n a p h th a len e-1-su lfon yla m in o)-1H-ben -
zoim id a zol-2-ylm eth yl]ben za m id in e Hyd r och lor id e (10).
Starting from 25b and naphthalene-1-sulfonyl chloride, the
title compound was synthesized following the procedures
described above. LRMS (EKA): (M + H)+ ) 470. 1H NMR
(DMSO-d6): δ 3.60 (s, 3H), 4.31 (s, 2H), 6.86 (dd, 1H), 7.11 (d,
1H), 7.27 (d, 1H), 7.40-7.81 (m, 7H), 8.00-8.20 (m, 3H), 8.79
(d, 1H), 9.19 (s, 2H), 9.35 (s, 2H), 10.48 (s, 1H). Anal.
(C26H23N5O2S‚HCl‚H2O) C, H, N.
4-(5-Ben zen esu lfon yla m in o-1-m eth yl-1H-ben zoim id a -
zol-2-ylm eth yl)ben za m id in e Hyd r och lor id e (2). Gaseous
anhydrous HCl was bubbled through a solution of nitrile 26b
(430 mg, 1.07 mmol) in 30 mL of cold ethanol (0 °C) for 0.5 h
and allowed to stir for 5 h at room temperature. The solvent
was evaporated in vacuo, and the residue was redissolved in
30 mL of ethanol. Ammonium carbonate (1.0 g, 10.7 mmol)
was added, and the reaction mixture was stirred overnight at
room temperature. Evaporation in vacuo afforded a solid mass
that was purified via chromatography (silica gel, dichlo-
romethane/methanol 5:1) to afford the title compound (39%)
as an amorphous solid. LRMS (EKA): (M + H)+ ) 420, (2M
+ H)+ ) 839. 1H NMR (DMSO-d6): δ 3.69 (s, 3H), 4.38 (s, 2H),
6.98 (dd, 1H), 7.22 (d, 1H), 7.37 (d, 1H), 7.50 (d, 2H), 7.41-
7.73 (m, 5H), 7.79 (d, 2H), 9.24 (s, 2H), 9.40 (s, 2H), 10.11 (s,
1H). Anal. (C22H21N5O2S‚HCl) C, H, N, Cl.
4-(5-Ben zen esu lfon yla m in o-1H -b en zoim id a zol-2-yl-
m eth yl)ben za m id in e Hyd r och lor id e (3). Starting from
4-nitro-benzene-1,2-diamine, the title compound was synthe-
sized following the procedures described above. LRMS
1
(EKA): (M + H)+ ) 406, (2M + H)+ ) 811. H NMR (DMSO-
4-[1-Meth yl-5-(n a p h th a len e-2-su lfon yla m in o)-1H-ben -
zoim id a zol-2-ylm eth yl]ben za m id in e Hyd r och lor id e (11).
Starting from 25b and naphthalene-2-sulfonyl chloride, the
title compound was synthesized following the procedures
described above. LRMS (EKA): (M + H)+ ) 470. 1H NMR
(DMSO-d6): δ 3.62 (s, 3H), 4.32 (s, 2H), 6.98 (dd, 1H), 7.27 (d,
1H), 7.32 (d, 1H), 7.46 (d, 2H), 7.53-7.82 (m, 5H), 7.98 (dd,
1H), 8.05 (d, 2H), 8.32 (d, 1H), 9.16 (s, 2H), 9.33 (s, 2H), 10.22
(s, 1H). Anal. (C26H23N5O2S‚HCl‚H2O) C, H, N.
4-[5-(Isoqu in olin e-5-su lfon yla m in o)-1-m eth yl-1H-ben -
zoim id a zol-2-ylm eth yl]ben za m id in e Hyd r och lor id e (12).
Starting from 25b and isoquinoline-5-sulfonyl chloride, the
title compound was synthesized following the procedures
described above. LRMS (EKA): (M + H)+ ) 471. 1H NMR
(DMSO-d6): δ 3.62 (s, 3H), 4.32 (s, 2H), 6.86 (dd, 1H), 7.12 (d,
1H), 7.27 (d, 1H), 7.45 (d, 2H), 7.65-7.84 (m, 3H), 8.38 (d, 1H),
8.46 (d, 1H), 8.56 (d, 1H), 8.70 (d, 1H), 9.23 (s, 2H), 9.37 (s,
2H), 9.42 (s, 1H), 10.61 (s, 1H). Anal. (C25H22N6O2S‚HCl) C,
H, N.
Qu in olin e-8-su lfon ic Acid [2-(4-Cya n o-ben zyl)-1-m eth -
yl-1H-ben zoim id a zol-5-yl]a m id e (27). Like described for
compound 26b, the title compound was prepared via sulfona-
tion of 25b with quinoline-8-sulfonic acid chloride. LRMS
(YEF): M+ ) 453. 1H NMR (DMSO-d6): δ 3.69 (s, 3H), 4.36
(s, 2H), 6.96 (dd, 1H), 7.22 (d, 1H), 7.38 (d, 1H), 7.46 (d, 2H),
7.63 (t, 1H), 7.75 (m, 1H), 7.77 (d, 2H), 8.22 (m, 2H), 8.51 (dd,
1H), 9.18 (m, 1H), 10.18 (s, 1H).
d6): δ 4.28 (s, 2H), 6.89 (dd, 1H), 7.20 (d, 1H), 7.30 (d, 1H),
7.40-7.73 (m, 8H), 7.80 (d, 2H), 9.19 (s, 2H), 9.35 (s, 2H), 10.10
(s, 1H). Anal. (C21H19N5O2S‚HCl) C, H, N, Cl.
4-(5-Ben zen esu lfon yla m in o-1-eth yl-1H-ben zoim id a zol-
2-ylm eth yl)ben za m id in e Hyd r och lor id e (4). Starting from
N1-ethyl-4-nitro-benzene-1,2-diamine, the title compound was
synthesized following the procedures described above. LRMS
1
(EKA): (M + H)+ ) 434, (2M + H)+ ) 867. H NMR (DMSO-
d6): δ 1.13 (t, 3H), 4.21 (q, 2H), 4.38 (s, 2H), 6.97 (dd, 1H),
7.24 (d, 1H), 7.40 (d, 1H), 7.42-7.75 (m, 7H), 7.78 (d, 2H), 9.19
(s, 2H), 9.38 (s, 2H), 10.14 (s, 1H). Anal. (C23H23N5O2S‚HCl)
C, H, N, Cl.
4-(5-Ben zen esu lfon yla m in o-1-n -p r op yl-1H -b en zoim i-
d a zol-2-ylm eth yl)ben za m id in e Hyd r och lor id e (5). Start-
ing from N1-n-propyl-4-nitro-benzene-1,2-diamine, the title
compound was synthesized following the procedures described
1
above. LRMS (EKA): (M + H)+ ) 448, (2M + H)+ ) 895. H
NMR (DMSO-d6): δ 0.81 (t, 3H), 1.60 (m, 2H), 4.10 (q, 2H),
4.35 (s, 2H), 6.95 (dd, 1H), 7.22 (d, 1H), 7.38 (d, 1H), 7.40-
7.75 (m, 7H), 7.79 (d, 2H), 9.20 (s, 2H), 9.31 (s, 2H), 10.00 (s,
1H). Anal. (C24H25N5O2S‚HCl) C, H, N, Cl.
4-[5-(Ben zen esu lfon yl-m eth yl-am in o)-1-m eth yl-1H-ben -
zoim id a zol-2-ylm eth yl]ben za m id in e Hyd r och lor id e (6).
Compound 26b (0.70 g, 1.74 mmol) and iodomethane (0.26 g,
1.80 mmol) were dissolved in 20 mL of acetone, and potassium
carbonate (0.70 g, 5 mmol) was added. After it was stirred at
room temperature for 5 h, the reaction mixture was concen-
trated in vacuo, and the residue was purified via chromatog-
raphy (silica gel, dichloromethane/methanol 50:1) to afford the
intermediate N-[2-(4-cyano-benzyl)-1-methyl-1H-benzoimida-
zol-5-yl]-N-methyl-benzenesulfonamide (0.61 g, 83%). This was
converted into the title compound following the procedure of
the Pinner reaction described above. LRMS (EKA): (M + H)+
4-[1-Meth yl-5-(qu in olin e-8-su lfon ylam in o)-1H-ben zoim -
id a zol-2-ylm eth yl]ben za m id in e Hyd r och lor id e (13). Fol-
lowing the Pinner reaction protocol described above, the title
compound was prepared from 27. LRMS (EKA): (M + H)+
)
471. 1H NMR (DMSO-d6): δ 3.60 (s, 3H), 4.28 (s, 2H), 6.89
(dd, 1H), 7.10 (d, 1H), 7.20 (d, 1H), 7.43 (d, 2H), 7.67 (t, 1H),
7.20-7.30 (m, 3H), 8.22 (dd, 2H), 8.51 (dd, 1H), 9.15 (s, 2H),
9.18 (m, 1H), 9.32 (s, 2H), 9.78 (s, 1H). Anal. (C25H22N6O2S‚
HCl) C, H, N, Cl.
1
) 434, (2M + H)+ ) 867. H NMR (DMSO-d6): δ 3.18 (s, 3H),
3.75 (s, 3H), 4.41 (s, 2H), 6.92 (dd, 1H), 7.18 (d, 1H), 7.42-
7.75 (m, 8H), 7.80 (d, 2H), 9.18 (s, 2H), 9.38 (s, 2H). Anal.
(C23H23N5O2S‚HCl) C, H, N, Cl.
4-[5-(Qu in olin e-8-su lfon yl-N-(ca r boxym eth yl)-a m in o)-
1-m eth yl-1H-ben zoim idazol-2-ylm eth yl]ben zam idin e (14).
Compound 27 (2.30 g, 5 mmol) and bromo-acetic acid ethyl
ester (0.92 g, 5.5 mmol) were dissolved in 50 mL of dimethyl
sulfoxide (DMSO), and potassium carbonate (2.8 g, 20 mmol)
was added. After it was stirred at room temperature for 5 h,
the mixture was diluted with water (150 mL) and the
precipitate was isolated and purified via chromatography
(silica gel, dichloromethane/methanol 75:1) to afford quinoline-
8-sulfonic acid [2-(4-cyano-benzyl)-1-methyl-1H-benzoimidazol-
5-yl]-N-(ethoxycarbonylmethyl)amide (43%). The nitrile group
4-[1-Meth yl-5-(p yr id in e-3-su lfon yla m in o)-1H-ben zoim -
id a zol-2-ylm eth yl]ben za m id in e Hyd r och lor id e (7). Start-
ing from 25b and pyridine-3-sulfonyl chloride, the title com-
pound was synthesized following the procedures described
1
above. LRMS (EKA): (M + H)+ ) 421, H NMR (DMSO-d6):
δ 3.69 (s, 3H), 4.37 (s, 2H), 6.95 (dd, 1H), 7.25 (d, 1H), 7.38 (d,
1H), 7.51 (d, 2H), 7.55 (m, 1H), 7.79 (d, 2H), 8.07 (m, 1H), 8.73
(m, 2H), 9.19 (s, 2H), 9.35 (s, 2H), 10.31 (s, 1H). Anal.
(C21H20N6O2S‚HCl‚H2O) C, H, N, Cl.

1764 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Hauel et al.
was converted into the amidine following the procedure
described above, and the resulting 4-[5-(quinoline-8-sulfonyl-
N-(ethoxycarbonylmethyl)-amino)-1-methyl-1H-benzoimidazol-
2-ylmethyl]benzamidine hydrochloride (58%) was isolated as
an amorphous powder. This intermediate (0.40 g, 0.67 mmol)
was dissolved in a solution of sodium hydroxide (0.13 g, 3.37
mmol) in 10 mL of water and 3 mL of ethanol and stirred at
room temperature for 2 h. The mixture was then diluted with
water (15 mL) and neutralized with 1 N HCl. The amorphous
precipitate was isolated, dissolved in 1 N NaOH, and neutral-
ized with 0.5 N HCl. The precipitate was isolated and washed
with water to afford the zwitterionic title compound (56%) as
a crystalline powder; mp 222-224 °C (decomposition). LRMS
(EKA): (M + H)+ ) 529, (M + Na)+ ) 551, (M + 2H)2+ ) 265,
4-{1-Me t h yl-5-[N -ca r b oxym e t h yl(q u in olin e -8-su lfo-
n yl)am in o]-1H-ben zoim idazol-2-ylm eth oxy}ben zam idin e
Dih yd r och lor id e (17). Starting from (4-cyano-phenoxy)acetic
acid and N1-methyl-4-nitro-benzene-1,2-diamine, the title
compound was synthesized following the procedures described
for 28c and 18. In the final step, the zwitterionic product was
dissolved in 2 N HCl and then concentrated in vacuo. The
residue was triturated with ether, filtered, and dried to afford
the dihydrochloride as a white solid. LRMS (EKA): (M + H)+
1
) 545, (M + Na)+ ) 567. H NMR (DMSO-d6): δ 3.90 (s, 3H),
5.07 (s, 2H), 5.76 (s, 2H), 7.09 (dd, 1H), 7.38 (d, 2H), 7.53 (dd,
1H), 7.58-7.75 (m, 2H), 7.80 (dd, 1H), 7.92 (d, 2H), 8.13 (d,
1H), 8.32 (d, 1H), 8.61 (dd, 1H), 9.18 (s, 2H), 9.20 (m, 1H),
9.39 (s, 2H). Anal. (C27H24N6O5S‚2HCl‚0.5H2O) C, H, N, Cl.
1
(M + H + Na)2+ ) 276, (M + 2Na)2+ ) 287. H NMR (DMSO-
[(4-Met h yla m in o-3-n it r o-b en zoyl)p h en yl-a m in o]a ce-
tic Acid Eth yl Ester (29a ). A solution of 4-methylamino-3-
nitro-benzoic acid (2.0 g, 10.2 mmol) in 20 mL of thionyl
chloride and two drops of dimethyl formamide (DMF) was
refluxed for 0.5 h and then concentrated in vacuo. The residue
was dissolved in 30 mL of THF and dropwise added to a
solution of phenylamino-acetic acid ethyl ester (1.82 g, 10.1
mmol) in 30 mL of THF and 3 mL of triethylamine at room
temperature. The mixture was kept at ambient temperature
overnight and then concentrated in vacuo. The residue was
chromatographed (silica gel, dichloromethane/ethanol 99:1) to
afford the title compound (2.0 g, 55%). 1H NMR (DMSO-d6):
δ 1.19 (t, 3H), 2.90 (d, 3H), 4.12 (q, 2H), 4.57 (s, 2H), 6.81 (d,
1H), 7.17-7.40 (m, 6H), 7.98 (d, 1H), 8.33 (q, 1H).
({2-[2-(4-Ca r b a m im id oyl-p h en yl)et h yl]-1-m et h yl-1H -
ben zoim idazole-5-car bon yl}ph en yl-am in o)acetic Acid Hy-
d r och lor id e (19). Starting from intermediate 29a and 3-(4-
cyano-phenyl)propionic acid, the title compound was prepared
in five steps following the procedures described for compounds
14 and 18. LRMS (EKA): (M + H)+ ) 456, (M + Na)+ ) 478,
(M + 2Na)2+ ) 250.6. 1H NMR (DMSO-d6): δ 3.30 (t, 2H), 3.43
(t, 2H), 3.86 (s, 3H), 4.55 (s, 2H), 7.10-7.30 (m, 5H), 7.38 (dd,
1H), 7.58 (d, 2H), 7.65-7.88 (m, 4H), 9.23 (s, 2H), 9.42 (s, 2H).
Anal. (C26H25N5O3‚HCl‚H2O) C, H, N.
{[2-(4-Ca r b a m im id oyl-p h en oxym et h yl)-1-m et h yl-1H -
ben zoim idazole-5-car bon yl]ph en yl-am in o}acetic Acid Hy-
d r och lor id e (20). Starting from intermediate 29a and (4-
cyano-phenoxy)acetic acid, the title compound was prepared
in five steps following the procedure described for compounds
14 and 18. LRMS (EKA): (M + H)+ ) 458, (M + Na)+ ) 480,
(M + 2Na)2+ ) 251.6. 1H NMR (DMSO-d6): δ 3.85 (s, 3H), 4.53
(s, 2H), 5.62 (s, 2H), 7.05-7.40 (m, 8H), 7.60 (d, 1H), 7.62 (s,
1H), 7.89 (d, 2H), 9.12 (s, 2H), 9.30 (s, 2H). Anal. (C25H23N5O4‚
HCl‚H2O) C, H, N.
d6 + 2HCl): δ 3.92 (s, 3H), 4.77 (s, 2H), 5.07 (s, 2H), 7.16 (dd,
1H), 7.50-7.95 (m, 8H), 8.18 (d, 1H), 8.35 (d, 1H), 8.64 (d, 1H),
9.21 (d, 1H). Anal. (C27H24N6O4S‚H2O) C, H, N.
4-[5-(Qu in olin e-8-su lfon yl-N-(ω-ca r boxy-n -p r op yl)a m i-
n o)-1-m e t h yl-1H -b e n zoim id a zol-2-ylm e t h yl]b e n za m i-
d in e (15). Following the procedure described for compound
14, 27 was converted in three steps into the zwitterionic title
compound; mp 223-225 °C (decomposition). LRMS (EKA): (M
+ H)+ ) 557, (M + Na)+ ) 579, (M + 2H)2+ ) 279, (M + H +
Na)2+ ) 290. 1H NMR (DMSO-d6): δ 1.52 (m, 2H), 2.04 (t, 2H),
3.66 (s, 3H), 4.12 (t, 2H), 4.30 (s, 2H), 6.65 (dd, 1H), 7.09 (d,
1H), 7.23 (d, 1H), 7.40-7.68 (m, 5H), 7.75 (m, 1H), 8.02 (d,
1H), 8.22 (d, 1H), 8.56 (dd, 1H), 9.19 (dd, 1H). Anal.
(C29H28N6O4S) C, H, N.
4-[(1-Meth yl-5-n itr o-1H-ben zoim idazol-2-ylm eth yl)am i-
n o]ben zon itr ile (28c). This intermediate was synthesized in
two steps on the analogy of the procedure described for
compound 25b, starting from N1-methyl-4-nitro-benzene-1,2-
diamine and (4-cyano-phenylamino)acetic acid. 1H NMR
(DMSO-d6): δ 3.90 (s, 3H), 4.74 (d, 2H), 6.85 (d, 2H), 7.39 (t,
1H), 7.50 (d, 2H), 7.80 (d, 1H), 8.19 (dd, 1H), 8.51 (d, 1H).
4-({1-Met h yl-5-[N-ca r b oxym et h yl(q u in olin e-8-su lfo-
n yl)am in o]-1H-ben zoim idazol-2-ylm eth yl}am in o)ben zam i-
d in e (18). This compound was prepared on the analogy of the
procedure described for compound 14. Compound 28c (0.8 g,
2.6 mmol) was reduced via hydrogenation in a mixture of 150
mL of methanol and 100 mL of dichloromethane. The resulting
amino derivative (0.69 g, 2.48 mmol) was sulfonated with
quinoline-8-sulfonyl chloride (0.57 g, 2.5 mmol) in 10 mL of
pyridine at room temperature to afford quinoline-8-sulfonic
acid {2-[(4-cyano-phenylamino)methyl]-1-methyl-1H-benzoimi-
dazol-5-yl}amide (1.0 g, 86%), which was alkylated with bromo-
acetic acid ethyl ester (0.38 g, 2.3 mmol) and potassium
carbonate (0.83 g, 6.0 mmol) in refluxing acetone (yield: 1.0
g, 85%). This cyano compound was converted into the amidine
following the Pinner reaction protocol described above to afford
4-({1-methyl-5-[N-ethoxycarbonylmethyl(quinoline-8-sulfonyl)-
amino]-1H-benzoimidazol-2-ylmethyl}amino)benzamidine hy-
drochloride as an amorphous solid (0.69 g, 65%), which gave,
after alkaline hydrolysis, the zwitterionic title compound (91%)
as white crystals; mp 249-250 °C (decomposition). LRMS
({2-[(4-Ca r ba m im id oyl-p h en yla m in o)m eth yl]-1-m eth -
yl-1H-b en zoim id a zole-5-ca r bon yl}p h en yl-a m in o)a cet ic
Acid Hyd r och lor id e (21). Starting from intermediate 29a
and (4-cyano-phenylamino)acetic acid, the title compound was
prepared in five steps following the procedure described for
compounds 14 and 18. LRMS (EKA): (M + H)+ ) 457, (M +
1
Na)+ ) 479, (M + 2Na)2+ ) 251. H NMR (DMSO-d6): δ 3.92
(s, 3H), 4.52 (s, 2H), 4.91 (s, 2H), 6.91 (d, 2H), 7.08-7.30 (m,
5H), 7.39 (dd, 1H), 7.63 (s, 1H), 7.65-7.80 (m, 3H), 8.88 (s,
2H), 9.08 (s, 2H). Anal. (C25H24N6O3‚HCl‚0.5 H2O) C, H, N.
(EKA): (M + H)+ ) 544, (M + Na)+ ) 566, (M + H + Na)2+
)
1
283.8, (M + 2Na)2+ ) 294.6. H NMR (DMSO-d6): δ 3.69 (s,
3H), 4.58 (s, 2H), 4.60 (d, 2H), 6.46 (dd, 1H), 6.72 (d, 2H), 7.10
(d, 1H), 7.38 (t, 1H), 7.40-7.60 (m, 4H), 7.73 (dd, 1H), 8.08 (d,
1H), 8.20 (d, 1H), 8.27 (s, 2H), 8.53 (dd, 1H), 9.19 (m, 1H),
11.55 (s, 2H). Anal. (C27H25N7O4S‚2H2O) C, H, N, S.
3-({2-[(4-Car bam im idoyl-ph en ylam in o)m eth yl]-1-m eth -
yl-1H-ben zoim id a zole-5-ca r bon yl}p h en yl-a m in o)p r op i-
on ic Acid (22). The title compound was prepared on the
analogy of the synthesis of compound 21. LRMS (EKA): (M +
H)+ ) 471, (M + H + Na)2+ ) 247, (M + 2Na)2+ ) 258. 1H
2
4-(2-{1-Meth yl-5-[N-ca r boxym eth yl(qu in olin e-8-su lfo-
n yl)a m in o]-1H -b en zoim id a zol-2-yl}et h yl)b en za m id in e
(16). Starting from 3-(4-cyano-phenyl)propionic acid and N1-
methyl-4-nitro-benzene-1,2-diamine, the title compound was
synthesized following the procedures described for 28c and 18;
NMR (DMSO-d6 + HCl): δ 2.57 (t, 2H), 3.99 (s, 3H), 4.08 (t,
2H), 5.04 (s, 2H), 6.95 (d, 2H), 7.10-7.33 (m, 5H), 7.44 (dd,
1H), 7.70 (s, 1H), 7.80 (d, 2H), 7.83 (d, 1H). Anal. (C26H26N6O3‚
H2O) C, H, N.
4-({2-[(4-Car bam im idoyl-ph en ylam in o)m eth yl]-1-m eth -
yl-1H-ben zoim id a zole-5-ca r bon yl}p h en yl-a m in o)bu tyr -
ic Acid Hyd r och lor id e (23). The title compound was pre-
pared on the analogy of the synthesis of compound 21. LRMS
(EKA): (M + H)+ ) 485, (M + H + Na)2+ ) 254, (M + 2H)2+
mp 246-247 °C (decomposition). LRMS (EKA): (M + H)+
)
543, (M + Na)+ ) 565, (2M + 3Na)3+ ) 385. 1H NMR (DMSO-
d6): δ 3.24 (s, 4H), 3.56 (s, 3H), 4.60 (s, 2H), 6.55 (dd, 1H),
7.07 (d, 1H), 7.31 (d, 1H), 7.40 (d, 2H), 7.47-7.62 (m, 3H), 7.73
(dd, 1H), 8.04 (dd, 1H), 8.19 (d, 1H), 8.52 (dd, 1H), 8.63 (s,
2H), 9.20 (m, 1H), 12.02 (s, 2H). Anal. (C28H26N6O4S) C, H, N.
1
) 243. H NMR (DMSO-d6): δ 1.75 (m, 2H), 2.29 (t, 2H), 3.90
(t, 2H), 3.92 (s, 3H), 4.91 (s, 2H), 6.90 (d, 2H), 7.08-7.30 (m,

Design of Novel Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1765
5H), 7.39 (dd, 1H), 7.58-7.80 (m, 4H), 8.82 (s, 2H), 9.04 (s,
2H). Anal. (C27H28N6O3‚HCl‚0.5 H2O) C, H, N, Cl.
fragment residues 55-65.20 Cocrystals were generated by
soaking crystals with mother liquor containing 1 mM inhibitor.
Data were collected on a MAR Research imaging plate (X-ray
Research, Hamburg, Germany) mounted on a Rigaku RU200
rotating anode generator and processed and scaled with
HKL.21 Model building and refinement were carried out with
MAIN22 and CNS.23 The X-ray structures of compounds 2 and
24 were deposited in the Brookhaven data bank. The accession
numbers are 1KTT and 1KTS, respectively.
Mea su r em en t of Th r om b in In h ibit ion . The thrombin
inhibitory effects (IC50) of the compounds were determined
with a commercially available chromogenic assay (Roche,
Mannheim, Germany). Human thrombin (Roche) (0.042 U/mL)
was preincubated for 10 min at 37 °C with 10 different
dilutions (concentration range of 0.003-100 µM) of the test
compounds dissolved in DMSO or with DMSO as control. Upon
addition of the preincubation mixture to the chromogenic
substrate, tosyl-glycyl-prolyl-arginine-4-nitranilide acetate, ni-
traniline is cleaved by thrombin and the increase in absorbance
at 405 nm, related to the free nitraniline, is measured in a
spectrophotometer (Spectramax, Molecular Devices, Sunny-
vale, CA). By plotting the absorbance at 405 nm vs the
concentration of the test compound, the concentration that
induced a 50% thrombin inhibition (IC50) was calculated. All
measurements were performed in duplicate, and the mean
values of both determinations are represented.
Mea su r em en t of th e a P TT. aPTT was measured in a
coagulometer (Biomatic B10, Sarstedt, Germany) using the
PTT reagent of Boehringer, Mannheim, Germany, according
to the manufacturer’s instruction as a measure for the anti-
coagulant effect of the respective compound. Bloodsamples
were collected in sodium citrate solution (final concentration
0.313%). Each native blood sample (0.1 mL) was pipetted into
a test tube prewarmed to 37 °C. The PTT reagent (0.1 mL)
was added, mixed, and incubated for exactly 3 min. Calcium
chloride solution (0.1 mL), prewarmed to 37 °C, was added in
order to activate the coagulation cascade, and the time (aPTT;
in seconds) was determined that elapsed from the addition of
calcium chloride to the onset of clotting.
3-[(4-Meth ylam in o-3-n itr o-ben zoyl)pyr idin -2-yl-am in o]-
p r op ion ic Acid Eth yl Ester (29d ). Starting from 4-methy-
lamino-3-nitro-benzoic acid and 3-(pyridin-2-ylamino)propionic
acid ethyl ester, the title compound was prepared following
the procedure described for the synthesis of intermediate 29a ;
mp 86-88 °C. 1H NMR (DMSO-d6): δ 1.11 (t, 3H), 2.65 (t, 2H),
2.90 (d, 3H), 3.97 (q, 2H), 4.18 (t, 2H), 6.82 (d, 1H), 7.08 (d,
1H), 7.21 (m, 1H), 7.31 (dd, 1H), 7.70 (dt, 1H), 7.92 (d, 1H),
8.37 (q, 1H), 8.44 (dd, 1H). Anal. (C18H20N4O5) C, H, N.
3-({2-[(4-Car bam im idoyl-ph en ylam in o)m eth yl]-1-m eth -
yl-1H -b en zoim id a zole-5-ca r b on yl}p yr id in -2-yl-a m in o)-
p r op ion ic Acid Eth yl Ester Hyd r och lor id e (30). Com-
pound 29d (16.7 g, 44.7 mmol) was hydrogenated (Pd on
charcoal, 10%) in 200 mL of methanol at room temperature to
afford 3-[(3-amino-4-methylamino-benzoyl)pyridin-2-yl-amino]-
propionic acid ethyl ester (10.0 g, 65%) after purification via
chromatography (silica gel, dichloromethane/methanol 30:1).
This intermediate (2.1 g, 6.1 mmol) was added to a solution of
(4-cyano-phenylamino)acetic acid imidazolide (7.3 mmol) in 50
mL of THF and refluxed for 24 h. The reaction mixture was
then concentrated in vacuo, and the residue was dissolved in
30 mL of glacial acid and heated under reflux for 1 h. The
solution was diluted with 100 mL of water and neutralized
with concentrated ammonium hydroxide. Extraction with ethyl
acetate and purification via chromatography (silica gel, dichlo-
romethane/methanol 40:1) gave 3-({2-[(4-cyano-phenylamino)-
methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}pyridin-2-yl-
amino)propionic acid ethyl ester (1.8 g, 61%), which was
converted into the title compound following the protocol of the
Pinner reaction described above; yield: 1.4 g, 71%. LRMS
(EKA): (M + H)+ ) 500, (M + H + Na)2+ ) 261.8, (M + 2H)2+
) 250.8. 1H NMR (DMSO-d6): δ 1.12 (t, 3H), 2.68 (t, 2H), 3.77
(s, 3H), 3.97 (q, 2H), 4.22 (t, 2H), 4.66 (d, 2H), 6.88 (m, 3H),
7.13 (m, 2H), 7.36-7.60 (m, 4H), 7.65 (d, 2H), 8.40 (m, 1H),
8.67 (s, 2H), 8.90 (s, 2H).
3-({2-[(4-Car bam im idoyl-ph en ylam in o)m eth yl]-1-m eth -
yl-1H -b en zoim id a zole-5-ca r b on yl}p yr id in -2-yl-a m in o)-
p r op ion ic Acid (24). Compound 30 (1.0 g, 1.86 mmol) was
added to a solution of sodium hydroxide (0.24 g, 6.0 mmol) in
20 mL of water and 10 mL of ethanol and kept at ambient
temperature for 2 h. The mixture was then diluted with 60
mL of water and neutralized with acetic acid. The precipitate
was isolated and washed with water and ether to afford the
zwitterionic title compound (0.8 g, 91%) as white crystals; mp
276-277 °C. LRMS (EKA): (M + H)+ ) 472, (M + H + Na)2+
Ack n ow led gm en t. We thank Ingrid Christ, J osef
Eiband, Herbert Fischbach, Monika Kink-Eiband, Vera
Koch, Michael Koehler, Angela Schmid, J ohanna Schur-
er, and Elisabeth Waldmann for their skillful technical
assistance.
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallogra-
phy of complexes of compounds 2 and 24 with human R-throm-
bin; data collection and refinement statistics. Combustion
analysis of all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
1
) 247.6, (M + 2H)2+ ) 236.7, (M + 2Na)2+ ) 258.6. H NMR
2
(DMSO-d6 + HCl): δ 2.67 (t, 2H), 4.01 (s, 3H), 4.17 (t, 2H),
5.07 (s, 2H), 6.97 (d, 2H), 7.28-7.40 (m, 2H), 7.45 (dd, 1H),
7.70-7.94 (m, 5H), 8.93 (m, 1H). Anal. (C25H25N7O3‚2H2O) C,
H, N.
Refer en ces
3-({2-[(4-{Am in o-[(E)-h exyloxyca r bon ylim in o]m eth yl}-
p h en yla m in o)m et h yl]-1-m et h yl-1H -b en zoim id a zole-5-
ca r bon yl}p yr id in -2-yl-a m in o)p r op ion ic Acid Eth yl Ester
(31). Compound 30 (1.0 g, 1.86 mmol) was dissolved in 50 mL
of THF and 10 mL of water. Potassium carbonate (0.83 g, 6.0
mmol) was added, and the mixture was stirred at room
temperature for 15 min. Then, n-hexyl chloroformate (0.31 g,
1.86 mmol) was added and stirring was continued for another
hour. The organic phase was separated, dried over anhydrous
sodium sulfate, and concentrated in vacuo. The residue was
chromatographed (silica gel, dichloromethane/methanol 19:1)
to afford the title compound as colorless crystals; mp 128-
129 °C; 0.61 g, 51%. LRMS (EKA): (M + H)+ ) 628, (M + H
+ Na)2+ ) 325.7, (M + 2H)2+ ) 314.7. 1H NMR (DMSO-d6): δ
0.86 (t, 3H), 1.12 (t, 3H), 1.29 (m, 6H), 1.58 (m, 2H), 2.68 (t,
2H), 3.77 (s, 3H), 3.97 (m, 4H), 4.22 (t, 2H), 4.60 (d, 2H), 6.75
(d, 2H), 6.88 (d, 1H), 6.95 (t, 1H), 7.12 (m, 2H), 7.40 (d, 1H),
7.47 (d, 1H), 7.53 (dt, 1H), 7.80 (d, 2H), 8.39 (dd, 1H), 8.50-
9.30 (bs, 2H). Anal. (C34H41N7O5) C, H, N.
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