Heterocyclic thrombin inhibitors. Part 2: Quinoxalinone derivatives as novel, potent antithrombotic agents

Article abstract of DOI:10.1016/S0960-894X(03)00443-8

Quinoxalinone derivatives as prototypes of dual thrombin and factor Xa inhibitors have been discovered. Nanomolar inhibition of both coagulation enzymes resulted in very potent antithrombotic activity in vitro.

Full text of DOI:10.1016/S0960-894X(03)00443-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2297–2302
Heterocyclic Thrombin Inhibitors. Part 2: Quinoxalinone
Derivatives as Novel, Potent Antithrombotic Agents
Uwe J. Ries,a,* Henning W. M. Priepke,a Norbert H. Hauel,a Sandra Handschuh,a
Gerhard Mihm,a Jean M. Stassen,b Wolfgang Wienenb and Herbert Nara
aDepartment of Chemical Research, Boehringer Ingelheim Pharma KG, Birkendorfer Straße 65, D-88397 Biberach/Riß, Germany
bDepartment of Biological Research, Boehringer Ingelheim Pharma KG, Birkendorfer Straße 65, D-88397 Biberach/Riß, Germany
Received 16 January 2003; revised 9 April 2003; accepted 24 April 2003
Abstract—Quinoxalinone derivatives as prototypes of dual thrombin and factor Xa inhibitors have been discovered. Nanomolar
inhibition of both coagulation enzymes resulted in very potent antithrombotic activity in vitro.
# 2003 Published by Elsevier Ltd.
Excessive uncontrolled activation of the hemostatic sys-
tem results in thromboembolic diseases, a major cause
of morbidity and mortality in our society. The central
role of the serine proteases factor Xa and thrombin in
hemostasis make them attractive targets for antith-
rombotic therapy. Factor Xa, factor Va, calcium and
phospholipids formthe prothrombinase complex, which
converts prothrombin into thrombin. Thrombin very
efficiently initiates fibrin formation, platelet aggregation
and activation of factors V and VIII. Selective inhibitors
of thrombin and factor Xa are expected to be ther-
apeutically useful in the treatment or prophylaxis of
thromboembolic diseases.1
describes their synthesis, biological properties and the
X-ray structure of a trypsin–inhibitor complex.
The synthesis of compound 16 as a representative
example of quinoxalinone derivatives containing cyclo-
propyl carboxamide or related functions is described in
Scheme 1. Starting from commercially available 1-(4-
chlorophenyl)-1-cyclopropane carboxylic acid (2) a
nitro group was selectively introduced into position 3.
Nucleophilic displacement of the chlorine by methyl-
amine yielded the nitroaniline derivative 3, which was
subjected to catalytic reduction to give 4. The a-keto-
acid 5 was prepared by a two-step oxidation process
3
starting from4-cyano-phenylalanine. Subsequently 5
As part of our efforts to design and develop orally active
coagulation inhibitors containing heterocyclic core
structures we identified amidinophenoxy-quinoline
derivatives as potent thrombin inhibitors2 in vitro. The
ethoxycarbonyl-cyclopentyl derivative 1 turned out to
be the most potent compound within this series and
inhibited thrombin in the lower nanomolar range (Fig.
1). In addition 1 was the only example which also
showed weak inhibition of factor Xa. The X-ray struc-
ture of 1 bound to thrombin revealed an unexpected
binding mode of this new type of serine protease inhi-
bitor. Based on this lead structure novel coagulation
inhibitors having quinoxalinone as the central template
have been designed. These compounds inhibited throm-
bin and factor Xa in the nanomolar range. This paper
was condensed with 4 in refluxing ethanol to give the
quinoxalinone derivative 6, which was coupled with
pyrrolidine using TBTU/NMP/DMF to generate the
corresponding amide 7. Using a Pinner reaction
*Corresponding author. Tel.: +49-7351-544535; fax: +49-7351-
547169; e-mail: uwe.ries@bc.boehringer-ingelheim.com
Figure 1. Structure and biological activity of lead compound 1.
0960-894X/03/$ - see front matter # 2003 Published by Elsevier Ltd.
doi:10.1016/S0960-894X(03)00443-8

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U. J. Ries et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2297–2302
Scheme 1. (a) HNO3, À30 ꢀC, 93%; (b) CH3NH2 (40% in H2O), 80 ꢀC, 5 h, 94%; (c) H2, Pd/C (10%), MeOH, 25 ꢀC, 86%; (d) (CF3CO)2O, 40 ꢀC,
12 h, 70%; (e) CF3COOH (70%), 24 h, 25 ꢀC, 86%; (f) Compound 4, EtOH, 80 ꢀC, 6 h, 88%; (g) pyrrolidine, TBTU, NMP, DMF, 12 h, 25 ꢀC, 52%;
(h) (1) HCl/EtOH, 25 ꢀC, 6 h; (2) (NH4)2CO3, EtOH, 25 ꢀC, 12 h, 33%.
sequence the cyano group was converted into benzami-
dine 16.
On the other hand the X-ray structure of a protein/
inhibitor complex of compound 1 bound to thrombin
unexpectedly showed an interaction of the cyclopentyl
moiety with a lipophilic surface of thrombin in front of
the S4-subsite and occupation of the S4-subsite by the
ethyl ester functionality. The observed weak binding
affinity of this compound to factor Xa suggested a pro-
ductive interaction of this compound with this enzyme,
presumably binding to it with the same unusual binding
orientation. We concluded that 1 may be the prototype
for the design of dual thrombin and factor Xa inhibi-
tors, which might show superior antithrombotic activity
relative to selective inhibitors.4
In order to synthesize cyclopropyl amine analogues the
carboxylic acid derivative 6 was treated with diphenyl-
phosphoryl azide in the presence of t-BuOK/t-BuOH
followed by acidic removal of the Boc-protecting group
to yield 8 (Scheme 2). This intermediate was subjected
to reductive amination with cyclopentanone followed by
acylation, Pinner reaction and saponification to give
compound 22.
Amidino-phenoxy quinoline derivatives like compound
1 have been identified as highly potent and selective
thrombin inhibitors.2 However, the antithrombotic effi-
cacy in vitro as well as the pharmacokinetic properties
in vivo turned out to be insufficient for therapeutic use.
In the first step of lead optimization the central quino-
line template was replaced by a quinoxalinone moiety,
which has a reduced lipophilicity due to the additional
Scheme 2. (a) (PhO)2P(O)N3, TEA, t-BuOH, KOt-Bu, 80 ꢀC, 3 h, 97%; (b) 6N HCl, dioxane, 25 ꢀC, 4 h, 69%; (c) cyclopentanone, NaBH3CN,
AcOH, THF, 25 ꢀC, 24 h, 92%; (d) EtOOCCH2COCl, TEA, THF, 25 ꢀC, 1 h, 82%; (e) (1) HCl/EtOH, 25 ꢀC, 6 h; (2) (NH4)2CO3, EtOH, 25 ꢀC,
12 h, 80%; (f) NaOH (1M), EtOH, 25 ꢀC, 2 h, 54%.

U. J. Ries et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2297–2302
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Table 1. Quinoxalinone- and quinoline-type inhibitors: polarity and antithrombotic activity
Compd
Thrombin inhibition
IC50, mM
Factor Xa
inhibition IC50, mM
aPTT ED200
(mM)
Log P
(octanol/water, pH 7.4)
Trypsin inhibition
IC50, mM
Plasmin inhibition
IC50, mM
10
11
0.01
0.006
10.6
12.1
0.43
0.94
0.5
1.7
0.41
2.4
75
20
amide group. This modification resulted in equipotent
enzyme inhibitors with improved prolongation of the
activated partial thromboplastin time (aPTT) as exem-
plified for 10 compared to its quinoline analogue 11
(Table 1). The higher antithrombotic activity of 10 cor-
responds well with the lower lipophilicity of 10 as indi-
cated by its lower log P value (octanol/water).
At the piperidine ring a methyl group can be introduced
at position 3 without loss of affinity versus both
enzymes (18). On the other hand, replacement of the
piperidine moiety by piperazine results in decreased
activity (19). Interestingly, the introduction of a hydroxy-
methyl group at the pyrrolidine position 2 had no
influence on the inhibition of thrombin, whereas factor
Xa affinity is tenfold reduced (20).
The X-ray structure of compound 1 complexed with
thrombin had clarified that the ester group interacts
with the S4-subsite.2 Two of the cyclopentyl methylene
groups point into the bulk solvent indicating that they
do not contribute to the enzyme affinity. Therefore we
modified this fragment by exchanging the cyclopentyl
moiety with cyclopropyl or a,a-dimethyl-methylene in
order to reduce the lipophilicity.
The X-ray structure of 17 in complex with trypsin, used
here as a factor Xa active site surrogate,6 is shown in
Figure 2.7 As anticipated the benzamidine P1 group
forms the well-established twin-twin saltbridge interac-
tion with Asp 189. The S2-pocket of the enzyme is
neatly occupied by the N-methyl group of the central
quinoxalinone scaffold. The hydrophobic S4-pocket and
the lipophilic groove in front of the S4-subsite are
occupied by the piperidine ring and the geminal dime-
thyl substituents, respectively. A superimposition of the
trypsin bound conformation of 17 onto the active site of
factor Xa6b (factor Xa coordinates taken fromPDB-
entry 1G2L) reveals an unfavorable steric repulsion of
the quinoxalinone N-methyl group with the Y99 OH
group and of the piperidine moiety with F174. After
energy minimisation8 of this starting geometry of the
complex, it appears that a shift of the quinoxalinone
scaffold and a small change of the position of the
piperdine ring does not suffice to remove the steric
clash. In addition, a change of the original positions of
residues Y99 and F174 is necessary to accommodate the
inhibitor in the factor Xa active site. The energy
required for this induced fit might explain the moderate
binding affinity of 17 to factor Xa.
As shown in Table 2 the cyclopropyl derivative 12 and
the corresponding dimethyl-methylene analogue 10
(Table 1) were equipotent. The removal of the S4 sub-
stituent resulted in an approximately 100-fold loss of
activity in the corresponding acid 13. In contrast, intro-
duction of several amide groups resulted in a remark-
able improvement of the in vitro activity. The inhibition
of thrombin was independent on the amide type: the
dimethyl amide 14 as well as the pyrrolidine and piper-
idine derivatives 15–18 showed enzyme inhibition in the
lower nanomolar range. Compounds 12 and 14–17 also
inhibited trypsin with similar potency than factor Xa,
but were quite selective versus plasmin.
Factor Xa activity is strongly dependent on monocyclic
groups like piperidine or pyrrolidine amides. This can
be explained by the nature of the S4-subsite in factor
Xa, which is formed by aromatic amino acids and pre-
ferably interacts with cyclic or (hetero)aromatic resi-
dues.5 The cyclopropyl derivative 16 inhibits factor Xa
with an IC50 of 84 nM and was the most potent example
within this series. The high in vitro potency and the
increased polarity of these compounds (e.g., 15: log
P=À1.0, 16: log P=À1.5) results in a remarkable
improvement of the antithrombotic activity in vitro.
Compound 16 doubles the aPTT at a concentration less
than 0.1 mM (aPTT: ED200=0.095 mM) and, in this
regard, seems to be one of the most potent antith-
rombotic agents in vitro reported so far.
Unfortunately, non of the compounds 14–20 could be
used for in vivo studies due to cardiovascular side effects
(e.g., hypotension) at therapeutical dosages.9 On the
other hand, it has been reported in the literature that
introduction of a negatively charged group into a ben-
zamidine derivative resulted in improved tolerability
and pharmacokinetics in vivo.10 Therefore, the carboxyl-
ate containing analogues 21 and 22 were designed and
synthesized. As indicated with 21, the introduction of a
negatively charged group at the pyrrolidine ring is
accompanied with a 10-fold reduced thrombin affinity
and a complete loss of factor Xa inhibition. On the

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U. J. Ries et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2297–2302
Table 2. Biological activity of quinoxalinone derivatives
Compd
12
R
Thrombin inhibition
IC50, mM
Factor Xa Inhibition
IC50, mM
aPTT ED200
(mM)
Trypsin
inhibitionaIC50, mM
Plasmin
inhibitiona IC50, mM
0.027
3.3
46
0.67
0.58
n.d.
69
13
>100
>10
n.d.
14
15
0.016
0.005
9.2
0.5
0.41
0.16
9.5
>100
0.32
53
16
17
0.008
0.008
0.084
0.4
0.095
0.25
0.31
2.2
66
60
18
19
0.012
0.068
0.59
31
0.12
0.71
n.d.
n.d.
n.d.
n.d.
20
21
0.013
0.19
1.4
0.21
0.91
0.85
2.4
72
68
>100
22
0.015
3.0
0.45
0.55
36
an.d.=not determined.
other hand, when the cyclopropyl carboxamide moiety
was replaced by an acylated cyclopentyl-aminocyclo-
propyl group, a potent thrombin inhibitor was obtained
(22), which showed an aPTT doubling at a concen-
tration of 0.45 mM.
administration to rats. However, in the pharmacody-
namic studies in rats, both compounds showed an
unfavorable profile, which seemed to be inadequate for
therapeutic use. Therefore, further research con-
centrated on the identification of novel heterocyclic
analogues with improved pharmacokinetic profiles and
oral activity. The results of these efforts will be pub-
lished in the near future.
In contrast to the cationic compounds 14–20 the zwit-
terionic analogues 21 and 22 were well tolerated after iv-

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References and Notes
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Crystals were grown fromsmall seed crystals of the ‘open’
crystal form14 in 1.7–1.9 M ammonium sulfate, pH 6. Cocrys-
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˚
group P212121, cell constants: 63.5, 69.1, 63.7 A; Data collec-
˚
tion statistics: resolution limits 20.0–1.70 A, total observations
76868, unique reflections 29802, completeness 95.6%, R merge
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ꢀ
˚
fromideal geometry: bond length 0.008 A , bond angles 1.32 ,
2
2
2
˚
˚
˚
temperature factors (A ): 22.5 A , rms bonded Bs 1.4 A , R
factor 18.9%, R free 21.1%.
Figure 2. (a) Crystal structure of 17 in trypsin; (b) Model of 17 in
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the MMFF94s force field13 using MMFF94 charges. During
the calculations the factor Xa binding site remained flexible in
order to allow possible induced fits. The factor Xa binding site
˚
was defined by including residues within a sphere of 7 A radius
around 17 in its trypsin bound position.
Based on potent and selective thrombin inhibitors cov-
ering a central quinoline template, novel coagulation
inhibitors have been designed and synthesized. These
quinoxalinone derivatives inhibited thrombin and factor
Xa in the nanomolar range and were very potent
antithrombotic agents in vitro.11 However, compounds
with promising in vitro activity suffered from low toler-
ability or unfavorable pharmacokinetics. Recent results
of our efforts to identify potent dual inhibitors of
thrombin and factor Xa useful for preclinical and clin-
ical studies will be reported in the near future.
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Haemost. 2001: Suppl. Abs. P758.
12. MOE Molecular Environment, version 2002.03; Chemical
Computing Group Inc.: Montreal, Canada, 2002.
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We thank Gunter Hebel, Peter Fink, Simpert Koßler,
Georg Schadler and Angela Schmid for their excellent
technical assistance and Dr. Peter Sieger and Monika
Wollrath for the determination of log P values.

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