Low molecular weight dual inhibitors of factor Xa and fibrinogen binding to GPIIb/IIIa with highly overlapped pharmacophores

Article abstract of DOI:10.1016/j.ejmech.2013.03.056

Dual antithrombotic agents acting as anticoagulants and aggregation inhibitors could have substantial advantages over currently prescribed combinations of antithrombotic drugs. Herein, we report compounds with moderate inhibitory activity for factor Xa an

Full text of DOI:10.1016/j.ejmech.2013.03.056

European Journal of Medicinal Chemistry 64 (2013) 302e313
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Low molecular weight dual inhibitors of factor Xa and fibrinogen
binding to GPIIb/IIIa with highly overlapped pharmacophores
*
ꢀ
ꢀ
Uros Trstenjak, Janez Ilas, Danijel Kikelj
ꢀ
ꢀ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
Dual antithrombotic agents acting as anticoagulants and aggregation inhibitors could have substantial
advantages over currently prescribed combinations of antithrombotic drugs. Herein, we report com-
pounds with moderate inhibitory activity for factor Xa and fibrinogen GPIIb/IIIa binding (both in the
micromolar range). These compounds resulted from our efforts to merge the pharmacophores of se-
lective factor Xa inhibitor rivaroxaban with a mimic of the Arg-Gly-Asp (RGD) sequence of fibrinogen to
obtain designed multiple ligands with potential antithrombotic activity. Resulting from this study, a
structurally novel class of submicromolar fibrinogen GPIIb/IIIa binding inhibitor bearing 1,2,4-oxadiazol-
5(4H)-one moiety is also described.
Received 21 February 2013
Received in revised form
24 March 2013
Accepted 26 March 2013
Available online 6 April 2013
Keywords:
Factor Xa
Platelet fibrinogen receptor
GPIIb/IIIa
Ó 2013 Elsevier Masson SAS. All rights reserved.
Designed multiple ligands
Anticoagulant
Antiaggregant
1. Introduction
ligands [11] possessing anticoagulant and antiagreggatory activities
in a single molecule. Such dual antithrombotic agents could have
Thrombotic complications such as myocardial infarction, stroke,
unstable angina pectoris, and pulmonary embolism are a major
cause of mortality and morbidity worldwide [1]. Due to diverse
therapeutic disadvantages of currently prescribed anticoagulant
and antiaggregatory drugs, there is a growing need for the devel-
opment of better antithrombotic agents that would upgrade or
replace the existing antithrombotic therapy. Over the past decade,
major progress in the prevention of thromboembolic disorders has
been made by the introduction of novel inhibitors of thrombin
[2,3], factor Xa [4,5] and tissue factor/factor VIIa inhibitors [6,7],
and platelet GPIIb/IIIa receptor antagonists [8]. As observed in
clinical practice, concomitant use of anticoagulants (e.g., warfarin
or heparin) and antiaggregatory drugs (e.g., acetylsalicylic acid or
ticlopidine), as well as combinations of thrombin or fXa inhibitors
with GPIIb/IIIa antagonists, can display synergistic effects [9,10].
These observations stimulated research toward designed multiple
substantial advantages over combination therapies, including more
predictable and less complex pharmacokinetics and pharmacody-
namics [11,12], that could render them the antithrombotic drugs of
the future.
Predominant strategies for the preparation of designed multiple
ligands (DMLs) include the joining of pharmacophores of selective
ligands by cleavable or non-cleavable linkers and the overlapping of
pharmacophores resulting in fused or merged DMLs [11,12]. The first
class of DMLs in the field of antithrombotics were thrombin/fXa and
thrombin/factor VIIa inhibitors [13]. This class was followed by dual
antiplatelet compounds and finally by anticoagulant/antiplatelet
compounds such as thrombin inhibitors/GPIIb/IIIa antagonists,
antithrombin III-mediated fXa inhibitors/GPIIb/IIIa antagonists, and
thrombin or fXa inhibitors with antiaggregatory activity based on
different mechanisms [14]. The main challenge in the development
of antithrombotic designed multiple ligands turned out to be the
adjustment of desired activity ratios at different targets along with
their ability to modulate seemingly unrelated targets and simulta-
neously possess selectivity over closely related targets [15e18].
In recent publications our group reported on successful prepa-
ration of low molecular weight peptidomimetic compounds pos-
sessing thrombin inhibitory and GPIIb/IIIa antagonistic activities
[15e17] that were designed by overlapping mimetics of the
thrombin inhibiting D-Phe-Pro-Arg motif and the Arg-Gly-Asp
Abbreviations: CDI, 1,10-carbonyldiimidazole; DIEA, diisopropylethylamine;
DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide; DMLs, designed
multiple ligands; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; fXa, factor
Xa; HOBt, N-hydroxybenzotriazole; RGD, Arg-Gly-Asp sequence; THF, tetrahydro-
furan; TFA, trifluoroacetic acid.
* Corresponding author. Tel.: þ386 1 4769561; fax: þ386 1 4258031.
E-mail address: danijel.kikelj@ffa.uni-lj.si (D. Kikelj).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.056

U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
303
(RGD) sequence responsible for binding of fibrinogen to GPIIb/IIIa
(Fig. 1).
morpholinone moiety, and a 2-aminothiazole replacing the 5-
chlorothiophene terminus, was constructed and docked into the
binding sites of fXa [23] and GPIIb/IIIa [24]. A comparison of docked
structures to those in crystal structures of rivaroxaban/fXa [23],
tirofiban/GPIIb/IIIa, L-739758/GPIIb/IIIa and eptifibatide/GPIIb/IIIa
complexes [24] indicated that our ligands adopted suitable con-
formations and showed promising interactions with both targets.
In the 4-carboxypiperidine series of compounds, we also explored
alternative arginine mimetics to the 2-aminothiazole group of
previous series, with the aims of optimally exploiting the interac-
tion with Asp189 in the S1 pocket of fXa, raising the GPIIb/IIIa
antagonistic activity, and balancing the activities against both tar-
gets. To this end, the 2-aminothiazole group was replaced with
benzamidine, 3-amino-4-chlorophenyl and 3-carbamimidoyl-4-
chlorophenyl moieties attached to the rest of the molecule via an
amide or methylenamino linkage.
Successful preparation of compounds possessing inhibitory ac-
tivity toward thrombin and fibrinogen-GPIIb/IIIa binding [15e18],
combined with a hypothesis that targeting fXa could be a more
effective strategy than targeting thrombin for anticoagulation [19e
22], led us to the idea of combining pharmacophores of fXa in-
hibitors and GPIIb/IIIa antagonists to yield a low molecular weight,
dual acting compound that would inhibit fXa and fibrinogen
binding to GPIIb/IIIa. During the course of our work, a report that
the antithrombotic activity of EP224283, which is a dual fXa in-
hibitor/GPIIb/IIIa antagonist prepared by linking fXa inhibitor
idraparinux and GPIIb/IIIa antagonist tirofiban with polyethyl
eneglycol, exceeded that of the coadministered parent compounds
[19], strenghtened our belief that targeting fXa and GPIIb/IIIa with
the envisaged dual acting compounds might be a viable concept in
antithrombotic therapy.
2.2. Chemistry
2. Results and discussion
The synthesis of target dual-activity compounds 7, 8, and 11e20
is depicted in Scheme 1. Both enantiomers of the key intermediate,
ethyl 1-(4-(5-(aminomethyl)-2-oxooxazolidin-3-yl)phenyl)piperi-
dine-4-carboxylate (5), were prepared by hydrazinolysis of the
phthalimido-protected precursor (4), which was synthesized in
three steps from ethyl 1-(4-nitrophenyl)piperidine-4-carboxylate
(1) by catalytic hydrogenation, opening of N-(2,3-epoxypropyl)
phthalimide by the resulting amine 2, and subsequent cyclization
of 3 to oxazolidin-2-one 4, as described previously [23]. Interme-
diate 5 was then coupled with tert-butyl (5-(chlorocarbonyl)thia-
zol-2-yl)carbamate, 4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)ben
zoic acid, 3-amino-4-chlorobenzoic acid, or 4-chloro-3-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)benzoic acid to give compounds 6, 9,
17, and 19, or subjected to reductive amination using 4-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)benzaldehyde to give compound 10.
Compounds 7, 8, 11, 12, 13, 14, 15, 16, 18, and 20 were then prepared
by removal of protecting groups employing trifluoroacetic acid-
mediated cleavage of tert-butyl carbamate, alkaline hydrolysis
of ethyl ester, and hydrogenolytic cleavage of 1,2,4-oxadiazol-
5(4H)-one. Compounds 7, 8, and 9e18 were prepared in both
enantiomeric forms, whereas only the S-isomer was obtained for
19 and 20. The synthesis of enantiomers was performed starting
from (S)- and (R)-N-(2,3-epoxypropyl)phthalimide with 99% e.e.
using non-racemizing conditions applied in the synthesis of rivar-
oxaban and standard non-racemizing coupling and deprotection
procedures.
2.1. Design
To prepare compounds combining in the same molecule the
capacities to inhibit both fXa and fibrinogen binding to GPIIb/IIIa,
we overlapped the pharmacophores of a direct fXa inhibitor
rivaroxaban and RGD sequence of fibrinogen (Fig. 2). The presence
of various P1 arginine-mimicking moieties in direct fXa inhibitors,
which contribute significantly to the potency of these compounds
via a direct electrostatic interaction with Asp189 in the S1 pocket of
fXa, suggested that the 5-chlorothiophene moiety of rivaroxaban
could be replaced by a 2-aminothiazole or benzamidine moiety
without significant effect on the inhibitory activity. The morpholi-
none moiety of rivaroxaban was replaced by piperidine carboxylate
moieties to find a substitution pattern that would be simultenously
well tolerated by the S4 pocket of fXa and provide a distance of 1.5e
1.8 nm between the introduced acidic and basic centers to enable
the binding of these modified rivaroxaban analogs to GPIIb/IIIa. The
rivaroxaban N-phenyl-(S)-oxazolidinone core was retained to allow
for an L-shape of the molecules, which is needed for directing
substituents into the S1 and S4 pockets of fXa, and to exploit two
hydrogen bonds to Gly219, which are observed with rivaroxaban
bound to fXa active site [23].
A small virtual library of ligands with (S) and (R) configurations
of the central oxazolidinone core, and containing a piperidine
carboxylate group in different positions as replacement for the
Fig. 1. Dual antithrombotic compound possessing well balanced inhibitory activities toward thrombin and fibrinogen-GPIIb/IIIa binding [17].

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U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
Fig. 2. Combining the pharmacophores of rivaroxaban (left) and the RGD sequence (middle).
Scheme 1. Reagents and conditions: (a) H2/PdeC, EtOH, rt, 10e15 h; (b) (S)-N-(2,3-epoxypropyl)phthalimide or (R)-N-(2,3-epoxypropyl)phthalimide, EtOH, reflux, 15 h; (c) CDI,
DMAP, THF, 60 ꢀC, 15 h; (d) hydrazine, EtOH, 80 ꢀC, 2 h; (e) tert-butyl (5-(chlorocarbonyl)thiazol-2-yl)carbamate, Et3N, DCM, 0 ꢀC / rt, 15 h; (f) TFA, DCM, 0 ꢀC / rt, 15 h; (g) LiOH,
MeOH/H2O ¼ 1:1, rt, 15 h; (h) 4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzoic acid, HOBt, EDC, DIEA, DMF, rt, 15 h for 9 or 4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)benzal-
dehyde, Na(OAc)3BH, DMF, rt, 15 h for 10; (i) H2/PdeC, CH3COOH, DMF, rt, 15 h; (j) 3-amino-4-chlorobenzoic acid, HOBt, EDC, DIEA, DMF, rt, 15 h; (k) 4-chloro-3-(5-oxo-4,5-dihydro-
1,2,4-oxadiazol-3-yl)benzoic acid, HOBt, EDC, DIEA, DMF, rt, 15 h.

U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
305
The analogs of compound 7a in which the 4-ethoxycarbonyl
piperidin-1-yl moiety was replaced by 3-ethoxycarbonylpiperidin-
1-yl group (i.e., 7b), 2-ethoxycarbonylpiperidin-1-yl group (7c), 4-
ethoxycarbonyl-3-oxopiperidin-1-yl group (7d), or 3-ethoxcar
bonyl-3-methyl-2-oxopiperidin-1-yl group (7e) (see Table 1 for
formulas) were prepared from (S)-N-(2,3-epoxypropyl)phthalimide
and respective ethyl 1-(4-nitrophenyl)piperidine carboxylates in
accordance to synthetic pathway 1 /8 to give a 1:1 mixture of two
diastereomers for each compound (see Supporting Information).
enable binding to GPIIb/IIIa. The determined activities of inhibition
of fXa, thrombin, and trypsin and of inhibition of fibrinogen binding
to GPIIb/IIIa for this series are given in Table 1. A penalty
for building GPIIb/IIIa binding activity into rivaroxaban was a sub-
stantial reduction in fXa inhibitory potency in the resulting com-
pounds. This effect was mainly due to replacement of the
2-chlorothiophene group with a 2-aminothiazole moiety because
2-chlorothiophene analogs of compounds 7aed and 8aee still
possessed nanomolar inhibitory constants for fXa [25]. Comparison
of fXa inhibitory potencies of ester/acid pairs 7a/8a, 7b/8b, 7f/8f
and ester 7c (the corresponding carboxylic acid could not be ob-
tained in pure form), which differ in the position of substitution on
the piperidine ring, showed only slight differences in the potencies
of the esters and their corresponding acids. Substitution at the
2.3. Biological evaluation
In the first series of compounds (Table 1), the 5-chlorothiophene
ring of rivaroxaban was replaced with the weakly basic
2-aminothiazole moiety and P4 morpholin-3-one with a piperidine
ring bearing a carboxylate group at positions 2, 3, or 4. 2-Amino
thiazole was expected to be well tolerated in the S1 subsite of fXa
because 2-chlorothiazole and 3-amino-2-chlorothiophene analogs
of rivaroxaban have displayed nanomolar potencies [23]. Our intent
was to find a P4 moiety that would provide suitable distance be-
tween its acidic group and the amidine motif of the 2-amino
thiazole moiety to mimic the RGD pharmacophore and thus
2-position (7c: Ki (fxa) ¼ 28.7 ꢁ 5.6
mM) proved to be most favorable.
In this case, the ethyl carboxylate group of phenylpiperidine-
2-carboxylate forces the two rings into a perpendicular arrange-
ment, thus mimicking perfectly the conformation of the
(3-oxomorpholine)phenyl moiety in rivaroxaban [23]. Moreover,
the same effect of the 2-oxo group can explain why 8e (Ki
¼ 11.2 ꢁ 2.3
m
M) displayed the most potent fXa inhibitory ac-
(fxa)
tivity in this series of compounds.
Table 1
Biological activity of P1 2-aminothiazole series of compounds: the inhibition of serine proteases fXa, thrombin and trypsin and the inhibition of fibrinogen binding to GPIIb/IIIa.
Comp.
R
Configuration (*)
R0
Ki (m
M)a
IC50 (m
M)b
FXa
Thrombin
Trypsin
GPIIb/IIIa
7a
S
Et
54.4 ꢁ 6.5
>100
>100
>300
8a
7f
8f
S
R
R
H
Et
H
48.8 ꢁ 4.0
111 ꢁ 19
>100
>100
>100
>100
>100
>100
78.4 ꢁ 23.7
>300
16.8 ꢁ 5.9
88.2 ꢁ 16.1
7b
S
Et
49.3 ꢁ 6.1
>100
>100
>300
8b
7c
S
S
H
47.5 ꢁ 7.5
28.7 ꢁ 5.6
>100
>100
>100
>100
>300
>300
Et
7d
S
Et
38.2 ꢁ 3.3
55.6 ꢁ 9.1
>100
>100
>100
>300
>300
7e
8e
S
S
Et
H
>100
11.2 ꢁ 2.3
0.66 ꢁ 0.09 nM
n.d.c
>100
>100
n.d.c
n.d.c
n.d.c
>300
Rivaroxaban
Tirofiban
Dabigatran
n.d.c
n.d.c
n.d.c
0.37 ꢁ 0.11 nM
n.d.c
6.3 ꢁ 1.1 nM
n.d.c
*Denotes the position of a stereogenic center in molecule.
a
Measurements were carried out in triplicate with three concentrations of the inhibitor and two concentrations of the substrate.
Assays were performed in duplicate and repeated at least three times on various days.
n.d.: not determined.
b
c

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U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
Compounds 7aef and 8aef showed no inhibition of the related
To fully exploit interaction with Asp189 in the S1 pocket of fXa,
increase the binding affinity for GPIIb/IIIa, and balance the activities
of compounds on both targets, in the second series of compounds
comprising a piperidine-4-carboxylic acid moiety (Table 2), 2-
aminothiazole was replaced either with a benzamidine group
linked to the rest of the molecule via an amide bond (compounds 13
and 15) or a methylenamino group (compounds 14 and 16), or with
a 3-amino-4-chlorophenyl moiety (compounds 17 and 18), while
our attempt to prepare a 3-carbamimidoyl-4-chlorophenyl deriv-
ative via intermediate 19 failed.
serine proteases thrombin and trypsin, whereas only the enantio-
mers 8a and 8f, which contain a piperidine-4-carboxylic acid
moiety, proved to be active both in the fXa inhibition assay and in
the GPIIb/IIIa binding inhibiton assay. The (R)-enantiomer 8f (IC50
¼ 16.8 ꢁ 5.9
m
M) showed 4.7-fold higher activity than the
(GPIIb/IIIa)
(S)-enantiomer 8a (IC50 (GPIIb/IIIa) ¼ 78.4 ꢁ 23.7
mM) for inhibition of
fibrinogen binding to GPIIb/IIIa, whereas 8f was 1.8-fold less potent
for inhibition of fXa than the (S)-enantiomer 8a (8f: Ki
¼ 88.2 ꢁ 16.1
m
M; 8a: Ki
¼ 48.8 ꢁ 4.0
mM). The better
(fxa)
(fxa)
binding observed for the (R)-enantiomer 8f to GPIIb/IIIa was in
agreement with our preliminary docking results, which indicated a
possible effect of stereochemistry on the binding of this series of
compounds to GPIIb/IIIa. The analogs of compounds 8a and 8f with
a 2-chlorothiophene group replacing the 2-aminothiazole moiety
possessed no fibrinogen GPIIb/IIIa binding inhibitory activity (IC50
Comparing the activities of the 2-aminothiazole derivative 8a
(Ki (fxa) ¼ 48.8 ꢁ 4.0
m
M; IC50 (GPIIb/IIIa) ¼ 78.4 ꢁ 23.7
m
M) with the
corresponding benzamidine compound 15a (Ki
¼
43.0
(fxa)
ꢁ 5.6
m
M; IC50 (GPIIb/IIIa) ¼ 12.0 ꢁ 1.0
mM) as well as those of 8f (Ki
¼ 88.2 ꢁ 16.1
¼ 62.9 ꢁ 9.2
m
M; IC50 (GPIIb/IIIa) ¼ 16.8 ꢁ 5.9
M; IC50
¼ 4.7 ꢁ 0.1
mM) and 15b (Ki
(fxa)
m
mM) lead to the
(fxa)
(GPIIb/IIIa)
> 300
mM; results not shown) which demonstrates the
conclusion that replacement of the 2-aminothiazole with a ben-
zamidine moiety resulted in 3.6e5.7-fold improvement of GPIIb/
IIIa binding, whereas no improvement of fXa inhibitory activity was
observed. Moreover, introduction of a benzamidine group rendered
compounds 13ae16a nonselective because they gained trypsin
(GPIIb/IIIa)
importance of the 2-aminothiazole terminus for GPIIb/IIIa binding.
In the tested series, the piperidine-4-carboxylic acid moiety
endowed 8a and 8f with weak dual activity, which is most likely
because of an appropriate distance between acidic and basic cen-
ters in the molecule, and thus a decision was made to retain the
piperidine-4-carboxylate group while exploring 2-aminothiazole
surrogates in the next series.
inhibitory activity with Ki values in the range 2.2e17.8
pound 15b (Ki M; IC50
3.79 ꢁ 0.46
m
M. Com-
¼
m
¼
(trypsin)
(GPIIb/IIIa)
4.7 ꢁ 0.1
m
M) was identified as a well-balanced, low micromolar,
Table 2
Inhibition of serine protease fXa, thrombin, and trypsin, and inhibition of fibrinogen binding to GPIIb/IIIa by benzamidine-, 3-amino-4-chlorophenyl-, and (5-oxo-4,5-dihydro-
1,2,4-oxadiazol-3-yl)phenyl compounds 11e20.
Comp.
R1
R2
R3
X
Conf.
Ki (m
M)a
IC50 (m
M)b
FXa
Thrombin
Trypsin
GPIIb/IIIa
11a
H
H
CO
S
>100
>100
>100
16.0 ꢁ 1.0
11b
12a
12b
H
H
H
H
H
H
CO
CH2
CH2
R
S
R
>100
>100
94.8 ꢁ 11.3
>100
>100
>100
>100
>100
>100
0.87 ꢁ 0.07
0.89 ꢁ 0.01
0.26 ꢁ 0.01
13a
H
Et
CO
S
>100
>100
5.65 ꢁ 0.35
14.0 ꢁ 2.0
13b
14a
14b
H
H
H
Et
Et
Et
CO
CH2
CH2
R
S
R
>100
>100
>100
>100
3.06 ꢁ 0.34
2.17 ꢁ 0.31
6.59 ꢁ 0.55
96.0 ꢁ 10.0
55.0 ꢁ 5.0
35.0 ꢁ 8.0
34.3 ꢁ 7.7
25.6 ꢁ 6.0
15a
H
H
CO
S
43.0 ꢁ 5.6
>100
8.95 ꢁ 0.77
12.0 ꢁ 1.0
15b
16a
17a
17b
18a
18b
H
H
NH2
NH2
NH2
NH2
H
H
Et
Et
H
H
CO
CH2
CO
CO
CO
CO
R
S
S
R
S
R
62.9 ꢁ 9.2
54.1 ꢁ 6.1
10.8 ꢁ 2.0
82.3 ꢁ 6.4
37.0 ꢁ 2.7
57.7 ꢁ 8.6
>100
3.79 ꢁ 0.46
17.8 ꢁ 1.2
>100
>100
>100
4.7 ꢁ 0.1
2.2 ꢁ 0.2
>300
53.0 ꢁ 4.6
Cl
>100
>100
>100
>100
>300
25.8 ꢁ 8.1
>100
12.8 ꢁ 1.0
20
H
CO
S
89.7 ꢁ 8.2
>100
>100
15.3 ꢁ 1.1
a
Measurements were carried out in triplicate with three concentrations of the inhibitor and two concentrations of the substrate.
Assays were performed in duplicate and repeated at least three times on various days.
b

U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
307
dual trypsin inhibitor/fibrinogen GPIIb/IIIa binding inhibitor for
which no therapeutic relevance has yet been identified. Replace-
ment of the amide bond in 15a with its reduced methylenamino
surrogate (16a) [26] resulted in a further 5.5-fold improvement
of GPIIb/IIIa binding with
fXa inhibitory activity (16a: Ki
a
concomitant slight decrease in
¼ 54.1 ꢁ 6.1 M; IC50
m
(fxa)
(GPIIb/
¼ 2.2 ꢁ 0.2
m
M). Ester analogs 14a and 14b showed a balanced
IIIa)
albeit moderate activity on both targets (e.g., 14b: Ki
¼
(fxa)
25.6 ꢁ 6.0
m
M; IC50 (GPIIb/IIIa) ¼ 35.0 ꢁ 8.0
mM).
Interestingly, intermediates 11be12b, which bear
a
1,2,4-
oxadiazol-5(4H)-one moiety as a masked amidino group, showed
submicromolar inhibition of fibrinogen binding to GPIIb/IIIa (11b:
IC50 (GPIIb/IIIa) ¼ 0.87 ꢁ 0.07
m
M; 12a: IC50 (GPIIb/IIIa) ¼ 0.89 ꢁ 0.01
mM;
12b: IC50 (GPIIb/IIIa) ¼ 0.26 ꢁ 0.01
m
M). These compounds present an
excellent starting point for optimization toward a structurally novel
class of nanomolar GPIIb/IIIa antagonists. Compounds 11be12b
were devoid of fXa inhibitory activity, as expected on the basis of
their structure.
Replacement of the 2-aminothiazole group with benzamidine
resulted only in improvement of fibrinogen GPIIb/IIIa binding in-
hibition, whereas fXa inhibitory activity remained almost un-
changed. Therefore, we hypothesized that introduction of
a
chlorine atom, which is a crucial P1 substituent for the fXa inhibi-
tory activity of rivaroxaban, onto the benzamidine moiety would
raise the fXa inhibitory activity of our compounds. Preparation
of the synthetically more tractable 3-amidino-4-chlorophenyl
analog was attempted but unfortunately failed in the last step, in
which the Pd-mediated hydrogenolytic cleavage of 1,2,4-oxadiazol-
5(4H)-one 20 to an amidino group resulted in partial removal of the
chlorine atom, and the low yield of this reaction prevented isolation
of a sufficient quantity of the 3-chloro-4-amidino analog. Again,
like the para-analog 11a, 1,2,4-oxadiazol-5(4H)-one 20 inhibited
the binding of fibrinogen to GPIIb/IIIa (IC50
¼
(GPIIb/IIIa)
15.3 ꢁ 1.1
mM) and showed no sustantial fXa inhibitory activity. In
the second attempt of introducing a chlorine atom, the benzami-
dine moiety of 15a and 15b was replaced with a 3-amino-4-
chlorophenyl moiety, which has been identified as a potent ben-
zamidine mimic [27] to obtain the enantiomers 18a (Ki
¼
(fxa)
37.0 ꢁ 2.7
m
M; IC50
¼ 25.8 ꢁ 8.1
m
mM).
M) and 18b (Ki
(GPIIb/IIIa)
¼ 57.7 ꢁ 8.6
mM; IC50 (GPIIb/IIIa) ¼ 12.8 ꢁ 1.0
(fxa)
Fig. 3. Compound 18a docked into the fXa active site (top) and GPIIb/IIIa active site
(bottom). The conformations of rivaroxaban in complex with fXa [23] and of tirofiban
in complex with GPIIb/IIIa [24] (from the X-ray crystal structures) are indicated in
green. The aIIb and b3 subunits of GPIIb/IIIa are shown in gray and blue. The figures
were prepared by Discovery Studio 3.1. (For interpretation of the references to color in
A comparison of the activities of enantiomeric pairs 8a/8f, 11a/
11b, 12a/12b, 14a/14b, 15a/15b, and 18a/18b demonstrates that (R)-
enantiomers are 2.6-folde18.4-fold more potent than (S)-enantio-
mers as inhibitors of fibrinogen binding to GPIIb/IIIa. This result is
in agreement with our preliminary docking results, whereas ste-
reochemistry does not display a consistent influence on fXa
inhibitory activity.
Fig. 3 displays binding configurations of compound 18a in the
active sites of fXa (PDB code: 2W26) and GPIIb/IIIa (PDB code:
2VDM) as predicted by CDOCKER, which is a CHARMM-based
docking program [28] that systematically positions the flexible li-
gands within a static active site and conducts low-level energy
calculations for each position. The 3-amino-4-chlorophenyl moiety
of compound 18a occupies the S1 subsite of fXa in which the po-
sition of the chlorine atom matches that of rivaroxaban and the
amino group forms a hydrogen bond to Asp189. Due to the bulki-
ness of the 3-amino-4-chlorophenyl moiety, the central oxazolidi-
none core of the molecule stretches over that of rivaroxaban. This
feature results in one hydrogen bond to Gly219 and influences the
improper occupation of the S4 subsite. The predicted binding
conformation of compound 18a to GPIIb/IIIa is extended and the
carboxylate group on the piperidine ring matches that of bound
tirofiban. It forms multiple hydrogen bonds to Tyr122, Ser123 and
this figure legend, the reader is referred to the web version of this article.)
with Tyr190 of the aIIb subunit, whereas the amino group of 3-
amino-4-chlorophenyl moiety fails to establish an expected
strong polar interaction with Asp224.
3. Conclusions
We describe the first low molecular weight, dual inhibitors of
fXa and fibrinogen binding to platelet fibrinogen receptor (GPIIb/
IIIa). These compounds were designed by overlapping the phar-
macophores of the selective fXa inhibitor rivaroxaban and the Arg-
Gly-Asp (RGD) sequence of fibrinogen. In the first series of com-
pounds, the 5-chlorothiophene ring of rivaroxaban was replaced
with a 2-aminothiazole ring, and the piperidine-4-carboxylate
moiety was identified as a morpholin-3-one replacement that is
well tolerated by the S4 pocket of fXa while simultaneously
enabling weak dual activity. In the next series of compounds,
which retain the piperidine-4-carboxylate group, the 2-amino
thiazole was replaced with benzamidine and 3-amino-4-chloro
phenyl moieties. These modifications led to compound 18a (Ki
Asn215 in the
b
3 subunit of the receptor while coordinating to the
proximal Mg2þ. The central phenyl ring forms a
pep
interaction

308
U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
¼ 37.0
m
M; IC50 (GPIIb/IIIa) ¼ 25.8
m
M) and 18b (Ki (fxa) ¼ 57.7
m
M;
1 atm H2 for 10e15 h at room temperature. The progress of the
reaction was monitored by TLC. After completion palladium on
activated charcoal was removed by filtration. Filtrate was evapo-
rated under reduced pressure yielding crude amine as redebrown
oil. Compounds 2aee were used without further purification.
(fxa)
IC50
¼ 12.8 M), which display balanced, moderate
m
(GPIIb/IIIa)
inhibitory activity for both targets and are selective against
thrombin and trypsin. As a part of these studies, compounds 11b,
12a and 12b bearing an 1,2,4-oxadiazol-5(4H)-one moiety were
identified as submicromolar inhibitors of fibrinogen binding to
GPIIb/IIIa that deserve further investigation. In conclusion, the
present study demonstrates that designing submicromolar dual
inhibitors of fXa and fibrinogen binding to GPIIb/IIIa remains a
demanding challenge due to constraints imposed by both targets.
4.1.3. General procedure foropeningof (S)- or(R)-N-(2,3-epoxypropyl)
phthalimides 3a, 3b, 3c, 3d, 3e, 3f
Suspension of starting compound (2aee) and (S)-N-(2,3-
epoxypropyl)phthalimide or (R)-N-(2,3-epoxypropyl)phthalimide
(1 eq.) in absolute ethanol (0.2 M) was refluxed for 15 h. The crude
product precipitated from clear solution at 0 ꢀC. Compounds 3aef
were purified either by flash column chromatography using EtOAc/
hexane as eluant or by recrystallization from absolute ethanol.
4. Experimental
4.1. Chemistry
Chemicals were obtained from Aldrich Chemical Co., Acros, and
Alfa Aesar and used without purification. Analytical TLC was per-
formed on silica gel Merck 60 F254 plates (0.25 mm), using visuali-
zation with ultraviolet light and ninhydrin. Column chromatography
was carried out on flash silica gel (particle size 40e240 mesh).
Melting points were determined on a Reichert hot stage microscope
and are uncorrected. Optical rotations were measured on a Perkine
Elmer 1241 MC polarimeter. The reported values for specific rotation
are average values of 10 successive measurements using an inte-
gration time of 5 s. 1H NMR spectra and 13C NMR spectra were
recorded on a Bruker AVANCE DPX300 spectrometer (1H NMR at
300.132 MHz and 13C NMR at 75.475 MHz) or Bruker AVANCE III
spectrometer (1H NMR at 400.130 MHz and 13C NMR at
100.613 MHz) in CDCl3 or DMSO-d6 solution. All chemical shift values
are reported in parts per million (ppm), the coupling constants (J) are
given in hertz, and the splitting patterns are appointed as: s (singlet),
d (doublet), dd (doublet of doublets), ddd (doublet of doublet of
doublets), td (triplet of doublets), t (triplet), dt (doublet of triplets),
and m (multiplet). Mass spectra were obtained using a VG Analytical
Autospec Q mass spectrometer. IR spectra were obtained on a Per-
kineElmer FTIR System Spectrum BX or Nicolet Nexus 470 FTIR
spectrometers. Microanalyses were performed on a PerkineElmer C,
H, N analyzer 240C. Analyses indicated by the symbols of the ele-
ments were within 0.4% of the theoretical values. HPLC analyses
were performed on an Agilent Technologies HP 1100 instrument
with a G1365B UVeVIS detector (254 nm), a G1316A thermostat, and
a G1313A autosampler, using an Agilent Eclipse Plus C18 column
4.1.4. General procedure for cyclization to oxazolidin-2-ones 4a, 4b,
4c, 4d, 4e, 4f
N,N0-Carbonyldiimidazole (2 eq.) and 4-(dimethylamino)pyri-
dine (catalytic amount) were added to a suspension of starting
compound (3aef) in tetrahydrofuran (0.1 M). The reaction mixture
was stirred for 15 h at 60 ꢀC. The crude product precipitated from
clear solution at 0 ꢀC. Compounds 4ae4f were purified either by
flash column chromatography using dichloromethane/methanol as
eluant or by recrystallization from absolute ethanol.
4.1.5. General procedure for preparation of 5a, 5b, 5c, 5d, 5e, 5f by
hydrazinolysis
To a solution of starting compound (4aef) in absolute ethanol
(0.1 M) hydrazine hydrate (55% solution) (3 eq.) was added. The
reaction mixture was stirred at 80 ꢀC for 2 h and afterward at 0 ꢀC
for 30 min. Precipitated white crystals of phthalhydrazide were
removed by filtration. Filtrate was concentrated under reduced
pressure yielding crystals of crude product. Compounds 5aef were
used without further purification.
4.1.6. General procedure for coupling of 5aef with tert-butyl (5-
(chlorocarbonyl)thiazol-2-yl)carbamate e synthesis of compounds
6a, 6b, 6c, 6d, 6e, 6f
Solution of tert-butyl (5-(chlorocarbonyl)thiazol-2-yl)carba-
mate (1.2 eq.) in dichloromethane (0.35 M) was added dropwise to
a stirred solution of starting compound (5aee) and triethylamine
(2 eq.) in dichloromethane (0.075 M) at 0 ꢀC. The reaction mixture
was stirred for 15 h at room temperature followed by removal of
solvent under reduced pressure to obtain a crude product, which
was purified by flash column chromatography using dichloro-
methane/methanol (19/1) or EtOAc as eluant.
(5
mm, 4.6 ꢂ 150 mm) at a flow rate of 1 mL/min. The eluant was a
mixture of 0.1% trifluoroacetic acid in water (A) and methanol (B).
The gradient was 80% A to 20% A over 15 min, then to 10% A over
1 min, then maintaining 10% A for 3 min, then to 80% A over 1 min.
4.1.1. Generalprocedurefor thepreparation ofethyl 1-(4-nitrophenyl)
piperidinecarboxylates 1a, 1b and 1c
4.1.7. General procedure for trifluoroacetic acid-mediated
deprotection of 6aef e synthesis of compounds 7a, 7b, 7c, 7d, 7e, 7f
To a solution of starting compound (6aef) in dichloromethane
(0.1 M) at 0 ꢀC trifluoroacetic acid (3 eq.) was added. The reaction
mixture was allowed to warm to room temperature and was stirred
for 15 h. Solvent and excessive trifluoroacetic acid were removed
under reduced pressure to obtain pure products 7aef.
To a stirred solution of ethyl isonipecotate (4.62 mL, 30 mmol) or
ethyl nipecotate (4.66 mL, 30 mmol) or ethyl pipecolinate (9.38 mL,
60 mmol) and 1-fluoro-4-nitrobenzene (1 eq.) in dimethylsulfoxide
(1 M) anhydrous potassium carbonate (1.5 eq.) was added. The
resulting mixture was stirred at 55 ꢀC for 15 h and after dilution
with cold water extracted with dichloromethane. The organic
phase was dried over Na2SO3, filtered and concentrated under
reduced pressure. Compound 1a was recrystallized from dichloro-
methane/hexane (1/19). Compounds 1b and 1c were isolated from
residue by flash column chromatography using dichloromethane or
hexane/EtOAc (5/1) as eluant.
4.1.8. General procedure for alkaline hydrolysis of ethyl esters and
subsequent trifluoroacetic acid-mediated deprotection of 6aef e
synthesis of compounds 8a, 8b, 8c, 8d, 8e, 8f
To a solution of starting compound (6aef) in methanol/water (1/
1) (0.05 M) lithium hydroxide (1.5 eq.) was added and the reaction
mixture was stirred at room temperature. The progress of the re-
action was monitored by TLC. After completion methanol was
removed under reduced pressure. Water phase was cooled to 0 ꢀC
and acidified (pH ¼ 1e2) with addition of conc. hydrochloric acid.
Precipitated crystals were separated by filtration, dried in vacuum
4.1.2. General procedure for catalytic hydrogenation e synthesis of
compounds 2a, 2b, 2c, 2d, and 2e
Mixture of starting compound (1aee) and 10% palladium on
activated charcoal in absolute ethanol (0.1 M) was stirred under

U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
309
and subjected to conditions of General procedure for trifluoroacetic
4.1.13. (S)-Ethyl 1-(4-(5-(aminomethyl)-2-oxooxazolidin-3-yl)
phenyl)piperidine-4-carboxylate (5a)
Crude product yield: 1.05 g (100%), white crystals. 1H NMR
(DMSO-d6)
1.27 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.81e1.94 (m, 2H,
acid-mediated deprotection of 6aef (4.1.7.). The crude products
were purified by flash column chromatography using dichloro-
methane/methanol (9/1) as eluant or used without purification.
d
CH2CHCH2), 2.00e2.05 (m, 2H, CH2CHCH2), 2.38e2.47 (m, 1H,
CH2CHCH2), 2.78 (dt, 2H, J ¼ 11.7 Hz, 2.9 Hz, CH2NCH2), 2.98 (dd,1H,
J ¼ 13.5 Hz, 5.8 Hz, CHCH2NH2), 3.08 (dd, 1H, J ¼ 13.6 Hz, 4.2 Hz,
CHCH2NH2), 3.58 (t, 1H, J ¼ 3.7 Hz, CH2NCH2), 3.61 (t, 1H, J ¼ 3.7 Hz,
CH2NCH2), 3.80 (dd, 1H, J ¼ 8.9 Hz, 6.6 Hz, ArNCH2CH), 4.00 (t, 1H,
J ¼ 8.9 Hz, ArNCH2CH), 4.08 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 4.60e4.68
(m, 1H, ArNCH2CH), 6.95 (d, 2H, J ¼ 9.2 Hz, AreH2,H6), 7.34 (d, 2H,
J ¼ 9.1 Hz, AreH3,H5), NH2 peak not seen. MS (ESI) (%) ¼ 348.2 (Mþ,
100), 118.1 (60), 77.0 (30). HRMS (ESI) calcd for C18H26N3O4
348.1923, found 348.1916.
4.1.9. Ethyl 1-(4-nitrophenyl)piperidine-4-carboxylate (1a)
Brown crystals of a crude product were recrystallized from
dichloromethane/hexane (1/19) to obtain yellow crystals, yield:
7.92 g (95%), mp 90e93 ꢀC. 1H NMR (CDCl3)
d
1.26 (t, 3H, J ¼ 7.2 Hz,
CH2CH3), 1.75e1.88 (m, 2H, CH2CHCH2), 1.99e2.06 (m, 2H,
CH2CHCH2), 2.53e2.57 (m,1H, CH2CHCH2), 3.06 (dd,1H, J ¼ 10.7 Hz,
2.7 Hz CH2NCH2), 3.10 (dd, 1H, J ¼ 10.7 Hz, 2.7 Hz CH2NCH2), 3.85 (t,
1H, J ¼ 3.6 Hz CH2NCH2), 3.90 (t, 1H, J ¼ 3.6 Hz CH2NCH2), 4.15 (q,
2H, J ¼ 7.1 Hz, CH2CH3), 6.81 (d, 2H, J ¼ 9.5 Hz, AreH2,H6), 8.09 (d,
2H, J ¼ 9.5 Hz, AreH3,H5). MS (ESI) (%) ¼ 279.1 (Mþ, 100). HRMS
(ESI) calcd for C14H19N2O4 279.1345, found 279.1348. IR (ATR) 2954,
2897, 1723, 1595, 1581, 1513, 1483, 1452, 1404, 1372, 1301, 1250,
4.1.14. (S)-Ethyl 1-(4-(5-((2-((tert-butoxycarbonyl)amino)thiazole-
5-carboxamido)methyl)-2-oxooxazolidin-3-yl)phenyl)piperidine-4-
carboxylate (6a)
1229, 1187, 1165, 1150, 1108, 1038, 1015 cmꢃ1
.
The crude product was purified by flash column chromatog-
raphy using dichloromethane/methanol (19/1) as eluant to give
white crystals, yield: 533 mg (48%), mp 269e272 ꢀC. 1H NMR
4.1.10. Ethyl 1-(4-aminophenyl)piperidine-4-carboxylate (2a)
Crude product yield: 9.30 g (100%), redebrown oil. 1H NMR
(CDCl3)
d
1.27 (t, 3H, J ¼ 7.2 Hz, CH2CH3), 1.82e2.04 (m, 4H,
(DMSO-d6)
d
1.19 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.50 (s, 9H, C(CH3)3),
CH2CHCH2), 2.32e2.42 (m, 1H, CH2CHCH2), 2.62e2.70 (m, 2H,
CH2NCH2), 3.40 (t, 1H, J ¼ 3.7 Hz CH2NCH2), 3.44 (t, 1H, J ¼ 3.7 Hz
CH2NCH2), 4.15 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 6.63 (d, 2H, J ¼ 8.8 Hz,
AreH2,H6), 6.81 (d, 2H, J ¼ 8.8 Hz, AreH3,H5), NH2 peak not seen. MS
(ESI) (%) ¼ 249.2 (Mþ, 100). HRMS (ESI) calcd for C14H21N2O2
249.1603, found 249.1599.
1.59e1.72 (m, 2H, CH2CHCH2), 1.90 (dd, 2H, J ¼ 13.2 Hz, 3.0 Hz,
CH2CHCH2), 2.41e2.46 (m, 1H, CH2CHCH2), 2.73 (dt, 2H, J ¼ 11.8 Hz,
2.5 Hz, CH2NCH2), 3.56e3.60 (m, 4H, CH2NCH2, CONHCH2), 3.78
(dd, 1H, J ¼ 9.0 Hz, 6.1 Hz, ArNCH2CH), 4.05e4.14 (m, 3H, CH2CH3,
ArNCH2CH), 4.74e4.82 (m, 1H, ArNCH2CH), 6.95 (d, 2H, J ¼ 9.1 Hz,
0
AreH2,H6), 7.35 (d, 2H, J ¼ 9.1 Hz, AreH3,H5), 8.00 (s, 1H, AreH4 ),
8.76 (t, 1H, J ¼ 5.7 Hz, CONHCH2), 11.71 (s, 1H, NHCOOteBu). MS
(ESI) (%) ¼ 574.2 (Mþ, 100), 474.2 (30), 259.6 (60). HRMS (ESI) calcd
for C27H36N5O7S 574.2335, found 574.2324. IR (ATR) 3289, 2932,
1742, 1716, 1623, 1543, 1518, 1433, 1407, 1367, 1307, 1272, 1246, 1216,
4.1.11. (S)-Ethyl 1-(4-((3-(1,3-dioxoisoindolin-2-yl)-2-hydroxypropyl)
amino)phenyl)piperidine-4-carboxylate (3a)
The crude product was recrystallized from absolute ethanol to
give beige crystals, yield: 2.79 g (55%), mp 148e150 ꢀC. [
a
]
20 þ7.3 (c
1164, 1142, 1095, 1045 cmꢃ1
.
D
0.25, DMSO). 1H NMR (CDCl3)
d
1.28 (t, 3H, J ¼ 7.1 Hz, CH2CH3),
1.83e2.05 (m, 4H, CH2CHCH2), 2.33e2.43 (m, 1H, CH2CHCH2), 2.65
(dd, 1H, J ¼ 10.6 Hz, 2.7 Hz, CH2NCH2), 2.69 (dd, 1H, J ¼ 10.8 Hz,
2.7 Hz, CH2NCH2), 3.15 (dd, 1H, J ¼ 13.2 Hz, 6.6 Hz, ArNHCH2), 3.27
(dd, 1H, J ¼ 13.2 Hz, 4.9 Hz, ArNHCH2), 3.42 (t, 1H, J ¼ 3.6 Hz,
CH2NCH2), 3.46 (t, 1H, J ¼ 3.6 Hz, CH2NCH2), 3.90e3.93 (m, 2H,
CON(CO)CH2CH), 4.23 (q, 3H, J ¼ 7.2 Hz, CH2CH3, CHOH), 6.66 (d,
2H, J ¼ 8.8 Hz, AreH2,H6), 6.86 (d, 2H, J ¼ 8.7 Hz, AreH3,H5), 7.74e
4.1.15. (S)-Ethyl 1-(4-(5-((2-aminothiazole-5-carboxamido)methyl)-
2-oxooxazolidin-3-yl)phenyl)piperidine-4-carboxylate (7a)
The crude product was purified by flash column chromatog-
raphy using dichloromethane/methanol (9/1) as eluant to give
20
white crystals, yield: 186 mg (72%), mp 110e113 ꢀC. [
a
]
ꢃ29.2 (c
D
0.21, DMSO). 1H NMR (DMSO-d6)
d
1.20 (t, 3H, J ¼ 7.1 Hz, CH2CH3),
1.62e1.69 (m, 2H, CH2CHCH2),1.96e2.00 (m, 2H, CH2CHCH2), 2.54e
2.60 (m, 1H, CH2CHCH2), 2.98 (t, 2H, J ¼ 11.2 Hz, CH2NCH2), 3.53e
3.61 (m, 4H, CH2NCH2, CONHCH2), 3.78 (dd, 1H, J ¼ 9.2 Hz, 6.2 Hz,
ArNCH2CH), 4.06e4.15 (m, 3H, CH2CH3, ArNCH2CH), 4.73e4.82 (m,
0
0
0
0
7.79 (m, 2H, AreH4 ,H7 ), 7.87e7.90 (m, 2H, AreH5 ,H6 ), NH and OH
peaks not seen. MS (ESI) (%) ¼ 452.0 (Mþ, 70), 406.0 (40), 261.0 (40),
248.0 (100). HRMS (ESI) calcd for C25H30N3O5 452.2185, found
452.2177. IR (KBr) 3324, 2944, 1772, 1715, 1516, 1466, 1429, 1395,
1308, 1253, 1172, 1090, 1030 cmꢃ1. Anal. calcd. for C25H29N3O5: C,
66.50; H, 6.47; N, 9.31; found C, 66.55; H, 6.39; N, 9.55.
1H, ArNCH2CH), 7.16 (d, 2H, J ¼ 8.6 Hz, AreH2,H6), 7.46 (d, 2H,
0
J ¼ 8.8 Hz, AreH3,H5), 7.75 (s, 1H, AreH4 ), 8.12 (brs, 2H, NH2), 8.62
(t, 1H, J ¼ 5.6 Hz, CONHCH2). 13C NMR (DMSO-d6)
d 14.05, 26.82,
42.00, 47.53, 50.50, 60.06, 71.36, 114.47, 117.38, 118.33, 119.35,
120.85, 137.03, 154.15,160.72,171.15,173.74, one alkyl C-atom behind
solvent peak. MS (ESI) (%) ¼ 474.2 (Mþ, 100), 237.6 (100), 214.6 (55),
200.6 (32). HRMS (ESI) calcd for C22H28N5O5S 474.1811, found
474.1828. IR (KBr) 3326, 3089, 2987, 1735, 1676, 1518, 1430, 1410,
1319, 1202, 1134, 1043 cmꢃ1. HPLC: 96.1%, tr ¼ 9.0 min.
4.1.12. (S)-Ethyl 1-(4-(5-((1,3-dioxoisoindolin-2-yl)methyl)-2-oxooxa
zolidin-3-yl)phenyl)piperidine-4-carboxylate (4a)
The crude product was recrystallized from absolute ethanol to
give beige crystals, yield: 1.44 g (49%), mp 183e188 ꢀC. [
a
]
20 ꢃ53.4
D
(c 0.25, DMSO). 1H NMR (CDCl3)
d
1.29 (t, 3H, J ¼ 7.1 Hz, CH2CH3),
1.82e2.07 (m, 4H, CH2CHCH2), 2.39e2.49 (m, 1H, CH2CHCH2), 2.78
(dt, 2H, J ¼ 11.8 Hz, 3.0 Hz, CH2NCH2), 3.58 (t, 1H, J ¼ 3.7 Hz,
CH2NCH2), 3.62 (t, 1H, J ¼ 3.7 Hz, CH2NCH2), 3.88 (dd, 1H, J ¼ 9.1 Hz,
5.9 Hz, ArNCH2CH), 3.98 (dd, 1H, J ¼ 14.0 Hz, 5.8 Hz, CON(CO)
CH2CH), 4.07e4.21 (m, 4H, CH2CH3, ArNCH2CH, CON(CO)CH2CH),
4.93e5.02 (m, 1H, ArNCH2CH), 6.94 (d, 2H, J ¼ 9.1 Hz, AreH2,H6),
4.1.16. (S)-1-(4-(5-((2-Aminothiazole-5-carboxamido)methyl)-2-
oxooxazolidin-3-yl)phenyl)piperidine-4-carboxylic acid (8a)
The crude product was purified by flash column chromatog-
raphy using dichloromethane/methanol (9/1) as eluant to give
20
white crystals, yield: 60 mg (39%), mp 275e280 ꢀC. [
a]
ꢃ34.0 (c
D
0
0
7.39 (d, 2H, J ¼ 9.1 Hz, AreH30 ,H5), 7.76e7.79 (m, 2H, AreH4 ,H7 ),
0.22, DMSO). 1H NMR (DMSO-d6)
d 1.63e1.76 (m, 2H, CH2CHCH2),
0
7.89e7.91 (m, 2H, AreH5 ,H6 ). MS (ESI) (%) ¼ 478.0 (Mþ, 100).
1.91e1.99 (m, 2H, CH2CHCH2), 2.39e2.45 (m, 1H, CH2CHCH2), 2.84
(t, 2H, J ¼ 10.4 Hz, CH2NCH2), 3.51e3.60 (m, 4H, CH2NCH2,
CONHCH2), 3.78 (dd, 1H, J ¼ 8.8 Hz, 6.0 Hz, ArNCH2CH), 4.11 (t, 1H,
J ¼ 8.8 Hz, ArNCH2CH), 4.71e4.80 (m, 1H, ArNCH2CH), 7.06 (d, 2H,
J ¼ 8.8 Hz, AreH2,H6), 7.41 (d, 2H, J ¼ 8.8 Hz, AreH3,H5), 7.68
HRMS (ESI) calcd for C26H28N3O6 478.1978, found 478.1981. IR (KBr)
3462, 2953, 2811, 1742, 1710, 1516, 1403, 1315, 1223, 1192, 1138,
1088, 1047 cmꢃ1. Anal. calcd. for C26H27N3O6: C, 65.40; H, 5.70; N,
8.80; found C, 65.17; H, 5.78; N, 8.96.

310
U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
0
(s þ brs, 3H, AreH4 , NH2), 8.49 (t, 1H, J ¼ 5.6 Hz, CONHCH2), 12.27
282 mg (41%). 1H NMR (DMSO-d6)
d
1.20 (t, 3H, J ¼ 7.1 Hz, CH2CH3),
(brs, 1H, COOH). 13C NMR (DMSO-d6)
d
27.38, 41.97, 47.67, 49.02,
1.66 (ddd, 2H, J ¼ 24.4 Hz, 11.3 Hz, 3.7 Hz, CH2CHCH2), 1.90 (dd, 2H,
J ¼ 13.2 Hz, 2.9 Hz, CH2CHCH2), 2.44e2.49 (m, 1H, CH2CHCH2), 2.72
(dt, 2H, J ¼ 11.9 Hz, 2.4 Hz, CH2NCH2), 3.58 (td, 2H, J ¼ 12.6 Hz,
3.3 Hz, CH2NCH2), 3.65 (t, 2H, J ¼ 5.6 Hz, CONHCH2), 3.81 (dd, 1H,
J ¼ 9.1 Hz, 6.1 Hz, ArNCH2CH), 4.06 (q, 2H, J ¼ 7.2 Hz, CH2CH3), 4.14
(t, 1H, J ¼ 9.1 Hz, ArNCH2CH), 4.79e4.86 (m, 1H, ArNCH2CH), 6.96
(d, 2H, J ¼ 9.2 Hz, AreH2,H6), 7.35 (d, 2H, J ¼ 9.2 Hz, AreH3,H5), 7.84
71.26, 116.80, 119.54, 120.90, 130.87, 141.25, 146.79, 154.23, 161.36,
171.62, 175.81, one alkyl C-atom behind solvent peak. MS (ESI)
(%) ¼ 446.1 (Mþ, 15), 85.1 (100). HRMS (ESI) calcd for C20H24N5O5S
446.1498, found 446.1482. IR (ATR) 3321, 3134, 1750, 1674, 1621,
1540,1515,1486,1454,1410,1388,1314,1190,1134,1079,1035 cmꢃ1
.
HPLC: 99.6%, tr ¼ 5.9 min.
0
0
(d, 1H, J ¼ 8.5 Hz, AreH5 ), 8.09 (dd, 1H, J ¼ 8.5 Hz, 2.2 Hz, AreH6 ),
0
4.1.17. General procedure for coupling of 5a and 5f with 4-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)benzoic acid (30), 3-amino-4-chloroben
zoic acid (31) or 4-chloro-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)
benzoic acid (34) e 9a, 9b, 17a, 17b, 19
8.19 (d, 1H, J ¼ 2.2 Hz, AreH2 ), 9.09 (t, 1H, J ¼ 5.7 Hz, CONHCH2),
NHCOON peak not seen. MS (ESI) (%) ¼ 570.2 (Mþ,100) for 35Cl, 572.2
(Mþ, 35) for 37Cl. HRMS (ESI) calcd for C27H29N5O7Cl 570.1756,
found 570.1745.
Diisopropylethylamine (2 eq.) and N0-(3-dimethylaminopropyl)-
N-ethylcarbodiimide (1.3 eq.) were added to a solution of starting
compound (5a or 5f), corresponding acid (1 eq.) and 1-hydroxy-1H-
benzotriazol-hydrate (1.3 eq.) in DMF (0.15 M according to the
starting compound). The reaction mixture was stirred for 15 h at
room temperature and then concentrated under reduced pressure.
The residue was cooled to 0 ꢀC and diluted with methanol. Precip-
itated crystals were separated by filtration to yield desired products.
4.1.21. General procedure for reductive amination of 5a and 5f e
synthesis of 10a, 10b
To a solution of starting compound (5a or 5f) and 4-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)benzaldehyde (1 eq.) in DMF (0.15 M)
Na(OAc)3BH (1.3 eq.) was added and the reaction mixture was
stirred for 15 h at room temperature. DMF was almost completely
removed under reduced pressure and the residue diluted with cold
water. Precipitated crystals were filtered off, washed with ether and
recrystallized from absolute ethanol. The product was not
completely soluble in hot ethanol.
4.1.18. (S)-Ethyl 1-(4-(2-oxo-5-((4-(5-oxo-4,5-dihydro-1,2,4-oxadia
zol-3-yl)benzamido)methyl)oxazolidin-3-yl)phenyl)piperidine-4-car
boxylate (9a)
Beige crystals, yield: 1.20 g (53%), mp 273e277 ꢀC. 1H NMR
4.1.22. (S)-Ethyl 1-(4-(2-oxo-5-(((4-(5-oxo-4,5-dihydro-1,2,4-oxadia
zol-3-yl)benzyl)amino)methyl)oxazolidin-3-yl)phenyl)piperidine-4-
carboxylate (10a)
(DMSO-d6)
d
1.20 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.61e1.71 (m, 2H,
CH2CHCH2), 1.88e1.92 (m, 2H, CH2CHCH2), 2.45e2.48 (m, 1H,
CH2CHCH2), 2.73 (dt, 2H, J ¼ 11.9 Hz, 2.4 Hz, CH2NCH2), 3.59 (td, 2H,
J ¼ 12.8 Hz, 3.5 Hz, CH2NCH2), 3.63e3.69 (m, 2H, CONHCH2), 3.83
(dd, 1H, J ¼ 8.9 Hz, 5.8 Hz, ArNCH2CH), 4.06e4.16 (m, 3H, CH2CH3,
ArNCH2CH), 4.81e4.87 (m, 1H, ArNCH2CH), 6.96 (d, 2H, J ¼ 9.1 Hz,
AreH2,H6), 7.36 (d, 2H, J ¼ 9.1 Hz, AreH3,H5), 7.91 (d, 2H, J ¼ 8.3 Hz,
Crude product yield: 212 mg (14%). 1H NMR (DMSO-d6)
d 1.20 (t,
3H, J ¼ 7.1 Hz, CH2CH3),1.61e1.71 (m, 2H, CH2CHCH2),1.88e1.92 (m,
2H, CH2CHCH2), 2.45e2.48 (m, 1H, CH2CHCH2), 2.73 (dt, 2H,
J ¼ 11.9 Hz, 2.4 Hz, CH2NCH2), 2.88 (d, 2H, J ¼ 5.4 Hz, NHCH2CH),
3.59 (td, 2H, J ¼ 12.8 Hz, 3.5 Hz, CH2NCH2), 3.78 (dd, 1H, J ¼ 8.9 Hz,
6.6 Hz, ArNCH2CH), 3.90 (s, 2H, ArCH2NH), 4.03e4.11 (m, 3H,
CH2CH3, ArNCH2CH), 4.71e4.78 (m, 1H, ArNCH2CH), 6.96 (d, 2H,
J ¼ 9.2 Hz, AreH2,H6), 7.38 (d, 2H, J ¼ 9.1 Hz, AreH3,H50), 7.55 (d, 2H,
0
0
0
0
AreH2 ,H6 ), 8.02 (d, 2H, J ¼ 8.3 Hz, AreH3 ,H5 ), 9.03 (t, 1H,
J ¼ 5.6 Hz, CONHCH2), 12.93 (brs, 1H, CNHCOO). MS (ESI)
(%) ¼ 536.2 (Mþ, 100). HRMS (ESI) calcd for C27H30N5O7 536.2145,
found 536.2149. IR (ATR) 3301, 3163, 1822, 1727, 1637, 1546, 1518,
0
0
0
J ¼ 8.3 Hz, AreH2 ,H6 ), 7.78 (d, 2H, J ¼ 8.3 Hz, AreH3 ,H5 ), NH and
NHCOON peaks not seen. MS (ESI) (%) ¼ 522.2 (Mþ, 100). HRMS (ESI)
calcd for C27H32N5O6 522.2353, found 522.2365. IR (ATR) 1807,
1723, 1653, 1517, 1479, 1432, 1412, 1388, 1309, 1221, 1174, 1145, 1105,
1476, 1435, 1415, 1386, 1309, 1227, 1171, 1140, 1044 cmꢃ1
.
4.1.19. (S)-Ethyl 1-(4-(5-((3-amino-4-chlorobenzamido)methyl)-2-
oxooxazolidin-3-yl)phenyl)piperidine-4-carboxylate (17a)
1042 cmꢃ1
.
Beige crystals, yield: 0.91 g (73%), mp 146e149 ꢀC. [
a
]
20 ꢃ33.8 (c
D
0.24, DMSO). 1H NMR (DMSO-d6)
d
1.20 (t, 3H, J ¼ 7.1 Hz, CH2CH3),
4.1.23. (S)-1-(4-(2-Oxo-5-((4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-
yl)benzamido)methyl)oxazolidin-3-yl)phenyl)piperidine-4-carboxylic
acid (11a)
Synthesized according to the alkaline hydrolysis part of the
General procedure for alkaline hydrolysis (4.1.8.). The crude prod-
uct (not completely soluble in hot methanol) was recrystallized
1.66 (ddd, 2H, J ¼ 24.4 Hz, 11.5 Hz, 3.7 Hz, CH2CHCH2), 1.90 (dd, 2H,
J ¼ 13.2 Hz, 3.1 Hz, CH2CHCH2), 2.44e2.49 (m, 1H, CH2CHCH2), 2.73
(dt, 2H, J ¼ 11.9 Hz, 2.5 Hz, CH2NCH2), 3.51e3.63 (m, 4H, CH2NCH2,
CONHCH2), 3.81 (dd, 1H, J ¼ 9.0 Hz, 5.9 Hz, ArNCH2CH), 4.06e4.13
(m, 3H, CH2CH3, ArNCH2CH), 4.76e4.83 (m, 1H, ArNCH2CH), 5.54
(brs, 2H, NH2), 6.96 (d, 2H, J ¼ 9.2 Hz, AreH2,H6), 7.00 (dd, 1H,
from methanol to obtain beige crystals, yield: 556 mg (73%), mp
0
0
J ¼ 8.3 Hz, 2.1 Hz, AreH6 ), 7.27 (d, 1H, J ¼ 8.4 Hz, AreH5 ), 7.28 (d,
269e272 ꢀC. [
a]
ꢃ43.9 (c 0.22, DMSO). 1H NMR (DMSO-d6)
20
D
0
1H, J ¼ 2.1 Hz, AreH2 ), 7.36 (d, 2H, J ¼ 9.1 Hz, AreH3,H5), 8.69 (t,1H,
d 1.59e1.69 (m, 2H, CH2CHCH2), 1.88e1.92 (m, 2H, CH2CHCH2),
J ¼ 5.8 Hz, CONHCH2). 13C NMR (DMSO-d6)
d
14.09, 27.44, 42.35,
2.34e2.40 (m, 1H, CH2CHCH2), 2.70 (dt, 2H, J ¼ 11.9 Hz, 2.4 Hz,
CH2NCH2), 3.59 (td, 2H, J ¼ 12.8 Hz, 3.5 Hz, CH2NCH2), 3.63e3.69
(m, 2H, CONHCH2), 3.83 (dd, 1H, J ¼ 8.9 Hz, 5.8 Hz, ArNCH2CH), 4.13
(t, 1H, J ¼ 8.9 Hz, ArNCH2CH), 4.81e4.87 (m, 1H, ArNCH2CH), 6.96
(d, 2H, J ¼ 9.1 Hz, AreH2,H6),07.36 (d, 2H, J ¼ 9.1 Hz, AreH3,H5), 7.91
47.76, 48.33, 59.87, 70.99, 114.52, 115.08, 116.30, 119.56, 119.77,
128.76, 130.10, 133.71, 144.62, 147.58, 154.26, 166.75, 174.22, one
alkyl C-atom behind solvent peak. MS (ESI) (%) ¼ 501.2 (Mþ, 100) for
35Cl, 503.2 (Mþ, 35) for 37Cl. HRMS (ESI) calcd for C25H30N4O5Cl
501.1905, found 501.1912. IR (ATR) 3353, 1739, 1649, 1614, 1573,
0
0
0
(d, 2H, J ¼ 8.3 Hz, AreH2 ,H6 ), 8.01 (d, 2H, J ¼ 8.3 Hz, AreH3 ,H5 ),
9.03 (t, 1H, J ¼ 5.5 Hz, CONHCH2), 12.23 (brs, 1H, CNHCOO), COOH
1538, 1512, 1489, 1429, 1392, 1301, 1258, 1226, 1137, 1087 cmꢃ1
.
HPLC: 100%, tr ¼ 19.3 min.
peak not seen. 13C NMR (DMSO-d6)
d 27.54, 42.48, 47.82, 48.46,
71.00, 116.27, 119.58, 126.04, 126.39, 128.08, 130.03, 136.87, 147.70,
154.26, 157.60, 160.85, 166.03, 175.92, one alkyl C-atom behind sol-
vent peak. MS (ESI) (%) ¼ 508.2 (Mþ, 100). HRMS (ESI) calcd for
4.1.20. (S)-Ethyl 1-(4-(5-((4-chloro-3-(5-oxo-4,5-dihydro-1,2,4-
oxadiazol-3-yl)benzamido)methyl)-2-oxooxazolidin-3-yl)phenyl)
piperidine-4-carboxylate (19)
C25H26N5O7 508.1832, found 508.1829. IR (ATR) 3300, 3149, 2955,
Crude product yield after purification by flash column chro-
matography using dichloromethane/methanol (19/1) as eluant:
2811, 1822, 1729, 1637, 1597, 1546, 1518, 1476, 1434, 1414, 1387, 1310,
1288, 1211, 1140, 1084, 1037, 1014 cmꢃ1. HPLC: 97.9%, tr ¼ 9.8 min.

U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
311
4.1.24. (S)-1-(4-(2-Oxo-5-(((4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-
yl)benzyl)amino)methyl)oxazolidin-3-yl)phenyl)piperidine-4-carboxy
lic acid (12a)
Synthesized according to the alkaline hydrolysis part of the
General procedure for alkaline hydrolysis (4.1.8.). The crude product
(not completely soluble in hot methanol) was recrystallized from
methanol to obtain white crystals, yield: 82 mg (25%), mp 177e
ArNCH2CH), 6.96 (d, 2H, J ¼ 9.2 Hz, AreH2,H6), 7.38 (d, 2H,
0
0
J ¼ 9.1 Hz, AreH3,H5),07.55 (d, 2H, J ¼ 8.3 Hz, AreH2 ,H6 ), 7.75 (d, 2H,
0
J ¼ 8.3 Hz, AreH3 ,H5 ), CNH(NH2) and NH peaks not seen. 13C NMR
(DMSO-d6) d 14.10, 24.52, 27.45, 47.93, 48.35, 51.05, 52.32, 59.88,
72.21, 116.31, 119.37, 127.47, 127.79, 130.27, 146.48, 147.48, 154.43,
165.62, 174.23, 176.22. MS (ESI) (%) ¼ 480.3 (Mþ, 20), 240.6 (100).
HRMS (ESI) calcd for C26H34N5O4 480.2611, found 480.2612. IR
(ATR) 2952, 1725, 1612, 1563, 1516, 1408, 1308, 1222, 1170, 1141,
1109, 1044, 1015 cmꢃ1. HPLC: 94.8%, tr ¼ 6.7 min.
181 ꢀC. [
a]
D
20 ꢃ30.7 (c 0.18, DMSO). 1H NMR (DMSO-d6)
d 1.59e1.69
(m, 2H, CH2CHCH2), 1.88e1.92 (m, 2H, CH2CHCH2), 2.34e2.42 (m,
1H, CH2CHCH2), 2.71 (dt, 2H, J ¼ 11.9 Hz, 2.3 Hz, CH2NCH2), 2.87 (d,
2H, J ¼ 5.4 Hz, NHCH2CH), 3.58 (td, 2H, J ¼ 12.5 Hz, 3.5 Hz, CH2NCH2),
3.78 (dd, 1H, J ¼ 8.9 Hz, 6.6 Hz, ArNCH2CH), 3.89 (s, 2H, ArCH2NH),
4.05 (t, 1H, J ¼ 8.9 Hz, ArNCH2CH), 4.71e4.78 (m, 1H, ArNCH2CH),
4.1.28. General procedure for alkaline hydrolysis of ethyl esters and
subsequent catalytic hydrogenation of 9a, 9b and 10a e synthesis of
compounds 15a, 15b, 16a
6.96 (d, 2H, J ¼ 9.2 Hz, AreH2,H60 ), 7.38 (d, 2H, J ¼ 9.1 Hz, AreH03,H50),
To a solution of starting compound (9a, 9b, 10a) in methanol/
water (1/1) (0.05 M) lithium hydroxide (1.5 eq.) was added and the
reaction mixture was stirred at room temperature. The progress of
the reaction was monitored by TLC. After completion methanol was
removed under reduced pressure. Water phase was cooled to 0 ꢀC
and acidified (pH ¼ 1e2) with addition of conc. hydrochloric acid.
Precipitated crystals were separated by filtration and subjected to
conditions of General procedure for catalytic hydrogenation of
1,2,4-oxadiazol-5(4H)-one (4.1.25.).
0
7.54 (d, 2H, J ¼ 8.3 Hz, AreH2 ,H6 ), 7.77 (d, 2H, J ¼ 8.3 Hz, AreH3 ,H5 ),
COOH, NH and NHCOON peaks not seen. 13C NMR (DMSO-d6)
d 27.54,
47.93, 48.48, 50.81, 52.18, 71.70, 116.28, 119.42, 123.48, 125.89,
128.80,130.15,143.39,147.60,154.32,159.16,162.55,175.96, one alkyl
C-atom behind solvent peak. MS (ESI) (%) ¼ 494.2 (Mþ, 100). HRMS
(ESI) calcd for C25H28N5O6 494.2040, found 494.2047. IR (ATR) 3302,
3075,1812,1729,1637, 1548,1516, 1478, 1414, 1386, 1308, 1223, 1139,
1043, 1015 cmꢃ1. HPLC: 96.4%, tr ¼ 6.9 min.
4.1.25. General procedure for catalytic hydrogenation of 1,2,4-oxadia
zol-5(4H)-one e synthesis of compounds 13a, 13b, 14a, 14b
4.1.29. (S)-1-(4-(5-((4-Carbamimidoylbenzamido)methyl)-2-oxooxa
zolidin-3-yl)phenyl)piperidine-4-carboxylic acid (15a)
Mixture of starting compound (9a, 9b, 10a, 10b), 10% of palla-
dium on activated charcoal and catalytic amount of DMF in glacial
acetic acid (0.05 M) was stirred under 1 atm H2 for 10e15 h at room
temperature. The progress of the reaction was monitored by TLC.
After completion palladium on activated charcoal was removed by
filtration. Filtrate was evaporated under reduced pressure and
product crystallized from ether or ether/methanol.
The crude product (not completely soluble in hot methanol) was
recrystallized from methanol to obtain beige crystals, yield: 286 mg
(69%), mp > 300 ꢀC. [
NMR (DMSO-d6)
a
]
D
20 ꢃ51.2 (c 0.23, 0.5% CF3COOH in DMSO). 1H
d
1.94e2.00 (m, 2H, CH2CHCH2), 2.13e2.17 (m, 2H,
CH2CHCH2), 2.63e2.71 (m, 1H, CH2CHCH2), 3.51 (t, 2H, J ¼ 11.0 Hz,
CH2NCH2), 3.59e3.62 (m, 2H, CH2NCH2), 3.68e3.70 (m, 2H,
CONHCH2), 3.92 (dd, 1H, J ¼ 9.0 Hz, 6.1 Hz, ArNCH2CH), 4.23 (t, 1H,
J ¼ 9.0 Hz, ArNCH2CH), 4.89e4.95 (m, 1H, ArNCH2CH), 7.61 (d, 2H,
J ¼ 9.0 Hz, AreH2,H6), 7.69 (d, 2H, J ¼ 9.1 Hz, AreH3,H5), 7.91 (d, 2H,
4.1.26. (S)-Ethyl 1-(4-(5-((4-carbamimidoylbenzamido)methyl)-2-
oxooxazolidin-3-yl)phenyl)piperidine-4-carboxylate (13a)
0
0
0
0
J ¼ 8.4 Hz, AreH2 ,H6 ), 8.04 (d, 2H, J ¼ 8.4 Hz, AreH3 ,H5 ), 9.14 (t,
The crude product was recrystallized from ether to obtain white
1H, J ¼ 5.6 Hz, CONHCH2), CNH(NH2) and COOH peaks not seen. 13
C
20
crystals, yield: 130 mg (63%), mp 197e200 ꢀC. [
a
]
ꢃ32.7 (c 0.25,
NMR (DMSO-d6) d 25.81, 36.90, 42.31, 47.38, 54.46, 71.46, 110.72,
D
DMSO). 1H NMR (DMSO-d6)
d
1.20 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.61e
113.59, 116.45, 118.91, 119.32, 121.80, 127.66, 128.25, 138.30, 154.06,
164.89, 165.69, 174.49. MS (ESI) (%) ¼ 466.2 (Mþ, 25), 233.6 (100).
HRMS (ESI) calcd for C24H28N5O5 466.2090, found 466.2084. IR
(ATR) 2943, 1736, 1658, 1544, 1513, 1484, 1407, 1381, 1285, 1230,
1206, 1140, 1108, 1041 cmꢃ1. HPLC: 98.3%, tr ¼ 5.9 min.
1.71 (m, 2H, CH2CHCH2), 1.76 (s, 3H, CH3COOH), 1.88e1.92 (m, 2H,
CH2CHCH2), 2.45e2.48 (m, 1H, CH2CHCH2), 2.73 (dt, 2H, J ¼ 11.9 Hz,
2.4 Hz, CH2NCH2), 3.59 (td, 2H, J ¼ 12.8 Hz, 3.5 Hz, CH2NCH2), 3.63e
3.69 (m, 2H, CONHCH2), 3.83 (dd, 1H, J ¼ 8.9 Hz, 5.8 Hz, ArNCH2CH),
4.06e4.16 (m, 3H, CH2CH3, ArNCH2CH), 4.81e4.87 (m, 1H,
ArNCH2CH), 6.96 (d, 2H, J ¼ 9.1 Hz, AreH2,H6), 7.36 (d, 2H,
4.1.30. (S)-1-(4-(5-(((4-Carbamimidoylbenzyl)amino)methyl)-2-
oxooxazolidin-3-yl)phenyl)piperidine-4-carboxylic acid (16a)
The crude product was recrystallized from ether/methanol to
0
0
J ¼ 9.1 Hz, AreH3,H5), 7.870(d, 2H, J ¼ 8.3 Hz, AreH2 ,H6 ), 8.01 (d,
0
2H, J ¼ 8.3 Hz, AreH3 ,H5 ), 9.09 (t, 1H, J ¼ 5.5 Hz, CONHCH2),
CNH(NH2) peaks not seen. 13C NMR (DMSO-d6)
d
14.10, 23.96, 27.44,
obtain beige crystals, yield: 30 mg (58%), mp 192e195 ꢀC. [
a]
20 ꢃ43.6
D
42.48, 47.77, 48.30, 59.88, 71.02, 116.28, 119.56, 127.61, 127.68,
130.05, 132.24, 137.73, 147.59, 154.26, 165.43, 165.89, 174.22, 175.56,
one alkyl C-atom behind solvent peak. MS (ESI) (%) ¼ 494.2 (Mþ, 25),
247.6 (100). HRMS (ESI) calcd for C26H32N5O5 494.2403, found
494.2404. IR (ATR) 2953, 1728, 1640, 1553, 1516, 1408, 1308, 1221,
1172, 1142, 1042, 1014 cmꢃ1. HPLC: 100%, tr ¼ 8.6 min.
(c 0.19, 0.5% CF3COOH in DMSO). 1H NMR (DMSO-d6)
d
1.59e1.69 (m,
2H, CH2CHCH2), 1.88e1.92 (m, 2H, CH2CHCH2), 2.34e2.42 (m, 3H,
CH2CHCH2, ArCH2NH), 2.66e2.71 (m, 2H, CH2NCH2), 3.20e3.22 (m,
2H, NHCH2CH), 3.54 (td, 2H, J ¼ 12.5 Hz, 3.5 Hz, CH2NCH2), 3.75 (dd,
1H, J ¼ 9.2 Hz, 6.7 Hz, ArNCH2CH), 4.10 (t,1H, J ¼ 9.1 Hz, ArNCH2CH),
4.83e4.90 (m, 1H, ArNCH2CH), 6.93 (d, 2H, J ¼ 9.3 Hz, AreH2,H6),
0
0
7.33 (d, 2H, J ¼ 9.2 Hz, AreH30 ,H5), 7.36 (d, 2H, J ¼ 8.2 Hz, AreH2 ,H6 ),
0
4.1.27. (S)-Ethyl 1-(4-(5-(((4-carbamimidoylbenzyl)amino)methyl)-
2-oxooxazolidin-3-yl)phenyl)piperidine-4-carboxylate (14a)
The crude product was recrystallized from ether/methanol to
7.68 (d, 2H, J ¼ 8.2 Hz, AreH3 ,H5 ), CNH(NH2), COOH and NH peaks not
seen. 13C NMR (DMSO-d6)
d 20.93, 25.95, 37.16, 41.53, 47.05, 54.00,
69.61, 110.78, 113.65, 116.51, 119.13, 119.38, 121.42, 128.02, 129.45,
144.42,153.51,165.20,171.93,174.62. IR (ATR) 3254, 2948,1741,1703,
1612, 1560, 1515, 1410, 1328, 1306, 1285, 1222, 1192,1169, 1135, 1042,
1005 cmꢃ1. HPLC: 100%, tr ¼ 6.0 min.
obtain bright orange crystals, yield: 44 mg (34%), mp 193e196 ꢀC.
20
[
a]
ꢃ23.9 (c 0.17, DMSO). 1H NMR (DMSO-d6)
d 1.20 (t, 3H,
D
J ¼ 7.1 Hz, CH2CH3), 1.61e1.72 (m, 2H, CH2CHCH2), 1.74 (s, 3H,
CH3COOH), 1.88e1.92 (m, 2H, CH2CHCH2), 2.45e2.48 (m, 1H,
CH2CHCH2), 2.73 (dt, 2H, J ¼ 11.9 Hz, 2.4 Hz, CH2NCH2), 2.78 (d, 2H,
J ¼ 5.2 Hz, NHCH2CH), 3.59 (td, 2H, J ¼ 12.8 Hz, 3.5 Hz, CH2NCH2),
3.78 (dd, 1H, J ¼ 8.9 Hz, 6.6 Hz, ArNCH2CH), 3.85 (s, 2H, ArCH2NH),
4.02e4.11 (m, 3H, CH2CH3, ArNCH2CH), 4.68e4.74 (m, 1H,
4.1.31. (S)-1-(4-(5-((3-Amino-4-chlorobenzamido)methyl)-2-oxooxa
zolidin-3-yl)phenyl)piperidine-4-carboxylic acid (18a)
Synthesized according to the alkaline hydrolysis part of the
General procedure for alkaline hydrolysis (4.1.8.). The crude

312
U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
product was recrystallized from ether/methanol to obtain beige
the presence and the absence of the inhibitor. Then, 50
buffer, the 50 M solution of each inhibitor concentration (or of
HBSA buffer in case of measurement without inhibitor), and 50
of enzyme solution were pipetted into the microtiter wells. The
plate was incubated for 15 min at 25 ꢀC, and 50
L of chromogenic
mM HBSA
20
crystals, yield: 149 mg (53%), mp 164e168 ꢀC. [
a
]
ꢃ21.7 (c 0.24,
m
D
DMSO). 1H NMR (DMSO-d6)
d
1.55e1.66 (m, 2H, CH2CHCH2), 1.81 (d,
mM
2H, J ¼ 11.3 Hz, CH2CHCH2), 1.90e1.95 (m, 1H, CH2CHCH2), 2.58 (t,
2H, J ¼ 11.2 Hz, CH2NCH2), 3.48e3.66 (m, 4H, CONHCH2, CH2NCH2),
3.88 (dd, 1H, J ¼ 8.4 Hz, 5.9 Hz, ArNCH2CH), 4.10 (t, 1H, J ¼ 8.9 Hz,
m
substrate was then added. The absorbance at 405 nm at 25 ꢀC was
measured every 10 s. Measurements were carried out in triplicate
with three concentrations of the inhibitor and two concentrations
of the substrate. For every combination of concentrations, Ki was
calculated from the change of absorbance in the initial, linear part
of the curve according to the method of Cheng and Prusoff [29],
and the final result was given as the average value. Rivaroxaban
(factor Xa, Ki ¼ 0.66 ꢁ 0.09 nM) and dabigatran (thrombin,
Ki ¼ 6.3 ꢁ 1.1 nM) were used as controls.
ArNCH2CH), 4.74e4.80 (m, 1H, ArNCH2CH), 5.58 (brs, 2H, NH2), 6.92
0
(d, 2H, J ¼ 8.9 Hz, AreH2,H6), 7.04 (d, 1H, J ¼ 8.2 Hz, AreH6 ), 7.25 (d,
0
2H, J ¼0 8.1 Hz, AreH5 ), 7.34 (d, 1H, J ¼ 9.1 Hz, AreH3,H5), 7.47 (s, 1H,
AreH2 ), 9.02 (t, 1H, J ¼ 5.4 Hz, CONHCH2), COOH peak not seen. 13
C
NMR (DMSO-d6)
d 29.12, 42.28, 43.55, 47.87, 49.24, 70.78, 114.72,
115.25, 115.99, 119.65, 128.70, 129.56, 133.58, 144.66, 148.29, 154.29,
166.68, 178.40, one alkyl C-atom behind solvent peak. MS (ESI)
(%) ¼ 473.2 (Mþ, 100) for 35Cl, 475.2 (Mþ, 30) for 37Cl. HRMS (ESI)
calcd for C23H26N4O5Cl 473.1592, found 473.1594. IR (ATR) 3310,
1740,1649,1538,1514,1488, 1409,1385,1315,1274,1208,1139, 1112,
1087 cmꢃ1. HPLC: 100%, tr ¼ 17.2 min.
4.2.2. Inhibition of in vitro binding of fibrinogen to isolated GPIIb/IIIa
receptors
Binding affinities to GPIIb/IIIa receptor were measured by a solid-
phase competitive displacement assay [31]. Human fibrinogen
(100 mg) was dissolved in aqueous NaCl (0.3 M, 5 mL) at 30 ꢀC and
then diluted with 0.1 M aqueous NaHCO3 to a final concentration of
1 mg/mL. Biotin N-hydroxysuccinimide ester (2 mg) was dissolved in
DMF (2 mL) and added to 6 mL of fibrinogen solution. The reaction
mixture was incubated for 90 min at 30 ꢀC and dialyzed for 3 h at
room temperature against buffer 1 (3 L, 20 mM Tris, 150 mM NaCl,
pH 7.4). After dialysis, the solution was centrifuged for 5 min at
5400 rpm, and Tween-20 (0.005%) was added to give the stock so-
4.1.32. (S)-1-(4-(5-((4-Chloro-3-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)benzamido)methyl)-2-oxooxazolidin-3-yl)phenyl)piperidine-4-
carboxylic acid (20)
Synthesized according to the alkaline hydrolysis part of the
General procedure for alkaline hydrolysis (4.1.8.). The crude prod-
uct (not completely soluble in hot methanol) was recrystallized
from methanol to obtain white crystals, yield: 168 mg (66%), mp
221e225 ꢀC. [
a
]
20 ꢃ38.9 (c 0.23, DMSO). 1H NMR (DMSO-d6)
d 1.64
D
(ddd, 2H, J ¼ 24.2 Hz, 11.4 Hz, 3.8 Hz, CH2CHCH2), 1.90 (dd, 2H,
J ¼ 13.2 Hz, 3.1 Hz, CH2CHCH2), 2.34e2.42 (m, 1H, CH2CHCH2), 2.72
(dt, 2H, J ¼ 11.9 Hz, 2.5 Hz, CH2NCH2), 3.58 (td, 2H, J ¼ 12.3 Hz,
3.2 Hz, CH2NCH2), 3.65 (t, 2H, J ¼ 5.5 Hz, CONHCH2), 3.82 (dd, 1H,
J ¼ 9.1 Hz, 6.1 Hz, ArNCH2CH), 4.14 (t, 1H, J ¼ 9.1 Hz, ArNCH2CH),
lution. Human integrin (10 mL of GPIIb/IIIa receptor solution (Cal-
biochem)) was diluted in 10.2 mL of buffer 2 (50 mM Tris, 150 mM
NaCl,1 mM CaCl2,1 mM MgCl2,1 mM MnCl2, pH 7.4) and adsorbed to
96-well (100
Lumitrac 600) overnight at 4 ꢀC. Nonspecific receptor binding sites
were blocked with 1% BSA in buffer 2 (200 L/well). Following in-
mL/well) high-binding microtiter plates (Greiner,
4.79e4.86 (m, 1H, ArNCH2CH), 6.96 (d, 2H, J ¼ 9.2 Hz, AreH2,H6),
0
7.36 (d, 2H, J ¼ 9.1 Hz, AreH3,H5), 7.805 (d, 1H, J ¼ 8.5 Hz, AreH5 ),
m
8.10 (dd, 1H, J ¼ 8.5 Hz, 2.2 Hz, AreH6 ), 8.20 (d, 1H, J ¼ 2.1 Hz, Are
cubation for 1 h at room temperature, the plates were washed twice
with buffer 3 (buffer 2 containing 0.1% Tween-20). The potential
antagonists were serially diluted with buffer 2 containing 0.1% of
0
H2 ), 9.09 (t,1H, J ¼ 5.8 Hz, CONHCH2),12.24 (brs,1H, NHCOO),13.00
(brs, 1H, COOH), COOH peak not seen. 13C NMR (DMSO-d6)
d 27.51,
42.53, 47.78, 48.50, 70.99, 116.31, 119.57, 123.00, 130.08, 130.40,
130.70, 131.97, 133.29, 135.02, 147.61, 154.23, 156.05, 159.43, 164.85,
175.93. MS (ESI) (%) ¼ 540.1 (Mþ, 100) for 35Cl, 542.1 (Mþ, 35) for
37Cl. HRMS (ESI) calcd for C25H23N5O7Cl 540.1286, found 540.1277.
IR (ATR) 3428, 3144, 1782, 1752, 1701, 1632, 1546, 1516, 1480, 1428,
1409,1388,1306,1288,1264,1223,1197,1172,1148,1121,1089,1042,
1026 cmꢃ1. HPLC: 100%, tr ¼ 18.1 min.
BSA and test solutions added (50
mL/well) together with biotinylated
fibrinogen (50 L/well, 1:42 dilution of stock solution in buffer 2
m
containing 0.1% of BSA) to each well. The plates were incubated for
2 h at room temperature and then washed twice with buffer 3.
Peroxidase-conjugated antibiotin goat antibody (100 mL/well of a
1:1000 dilution of purchased solution (Calbiochem) in buffer 2
containing 0.1% of BSA) was added to each well and incubated for
another hour. The microtiter plates were washed three times with
buffer 3. Finally, chemiluminescent substrate (POD, Roche Di-
4.2. Biochemical evaluation
agnostics, Boehringer Mannheim) (100 mL/well) was added, and the
4.2.1. Enzyme assay for inhibition of serine proteases
luminescence was measured with a BioTek Synergy H4 multimode
research reader three times over 10 min. Positive controls received
no inhibitors, whereas negative controls received no receptor. Tir-
ofiban (IC50 (GPIIb/IIIa) ¼ 0.37 ꢁ 0.11 nM) was used as the internal
standard. Assays were performed in duplicate and repeated at least
three times on various days. The mean experimental data were fitted
to the sigmoid model, and IC50 values were calculated from the
doseeresponse curve (OriginPro, OriginLab, version 7.5).
The enzyme amidolytic method for determining inhibition was
based on the spectrophotometric determination of absorbance of
the product formed after amide bond cleavage of a chromogenic
substrate in the presence of the enzyme. Ki, which is a quantitative
measure of inhibitor potency, was determined from the kinetics of
substrate hydrolysis with and without the addition of the inhibitor
[29,30]. Measurements (spectrophotometer, BioTek Synergy H4)
were performed in 96-well microtiter plates with a final volume of
200
mL (Km ¼ 2.6
and 40 M final concentration, and fXa at the final concentration of
m
L. Thrombin was tested at a final concentration of 0.5 NIH E/
4.3. Docking studies
mM) with the substrate S-2238 (Chromogenix) at 20
m
The binding modes for the ligands to fXa and GPIIb/IIIa were
studied by CDOCKER [28], a docking tool based on the CHARMm
force field, which is incorporated into Discovery Studio 3.1 (Accel-
rys Software Inc.). In CDOCKER, random ligand conformations were
generated through molecular dynamics, and a variable number of
translations/rotations were applied to each conformation to
generate low-energy orientations of the ligand within the active
site of rigid receptor. The orientations were further refined by grid-
1 mBAEE E/mL (Km ¼ 164
m
M) with the substrate S-2222 (Chro-
mogenix) at 100 and 200
mM final concentrations. Trypsin was
assayed at a final concentration of 0.5 nkat/mL through the use of
the substrate S-2222 (Chromogenix) at 50 and 100 M final con-
m
centrations. Inhibitors were dissolved in DMSO (concentration of
stock solutions, 10 mM) and diluted with distilled water to con-
centrations from 0.5 to 100 mM. Reaction rates were measured in

U. Trstenjak et al. / European Journal of Medicinal Chemistry 64 (2013) 302e313
313
[13] A. Kranjc, D. Kikelj, Dual inhibitors of the blood coagulation enzymes, Curr.
based simulated annealing (in our example 2000 heating steps,
target temperature 700 K, 5000 cooling steps, target temperature
300 K), which was followed by final minimization using full po-
tential. Final ligand conformations were sorted by CHARMm energy
(interaction energy plus ligand strain). The crystal structures of fXa
Med. Chem. 11 (2004) 2535e2547.
ꢂ
ꢀ
[14] M. Ilic, D. Kikelj, J. Ilas, Multitarget antithrombotic drugs, Curr. Top. Med.
Chem. 11 (2011) 2834e2848.
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[15] J. Ilas, Z. Jakopin, T. Borstnar, M. Stegnar, D. Kikelj, 3,4-Dihydro-2H-1,4-
benzoxazine derivatives combining thrombin inhibitory and glycoprotein
IIb/IIIa receptor antagonistic activity as a novel class of antithrombotic com-
pounds with dual action, J. Med. Chem. 51 (2008) 5617e5629.
ꢁ
(PDB entry code: 2W26, resolution 2.08 A) [23] and GPIIb/IIIa (PDB
ꢁ
ꢀ
ꢀ
ꢀ
[16] P. Stefanic Anderluh, M. Anderluh, J. Ilas, J. Mravljak, M. Sollner Dolenc,
M. Stegnar, D. Kikelj, Toward a novel class of antithrombotic compounds with
dual function. Discovery of 1,4-benzoxazine-3(4H)-one derivatives possessing
thrombin inhibitory and fibrinogen receptor antagonistic activities, J. Med.
Chem. 48 (2005) 3110e3113.
entry code: 2VDM, resolution 1.23 A) [24] were extracted from the
Brookhaven Protein Database. All ligands were docked in all
possible stereoisomeric forms in an active site located sphere with
ꢁ
ꢁ
8.62303 A for fXa and 11.72740 A radius for GPIIb/IIIa, which was
generated with the CreateSphere function around the subsequently
removed crystal structure ligand. A total of 100 dockings for each
ligand were performed, and the conformers with the lowest
CHARMm energy were chosen for interpreting the docking results.
ꢂ
ꢀ
[17] M. Ilic, D. Kikelj, J. Ilas, Fluorinated dual antithrombotic compounds based on
1,4-benzoxazine scaffold, Eur. J. Med. Chem. 50 (2012) 255e263.
ꢂ
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[18] M. Ilic, P. Dunkel, J. Ilas, E. Chabielska, A. Zakrzeska, P. Mátyus, D. Kikelj, To-
wards dual antithrombotic compounds balancing thrombin inhibitory
e
and fibrinogen GPIIb/IIIa binding inhibitory activities of 2,3-dihydro-1,4-
benzodioxine derivatives through regio- and stereoisomerism, Eur. J. Med.
Chem. 62 (2013) 329e340.
[19] B. Hechler, M. Freund, G. Alame, C. Leguay, S. Gaertner, J.P. Cazenave,
M. Petitou, C. Gachet, The antithrombotic activity of EP224283, a neutralizable
dual factor Xa inhibitor/glycoprotein IIbIIIa antagonist, exceeds that of
the coadministered parent compounds, J. Pharmacol. Exp. Ther. 338 (2011)
412e420.
[20] A. Turpie, Oral, direct factor Xa inhibitors in development for the prevention
and treatment of thromboembolic diseases, Arterioscler. Thromb. Vasc. Biol.
27 (2007) 1238e1247.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.03.056.
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