Towards dual antithrombotic compounds-Balancing thrombin inhibitory and fibrinogen GPIIb/IIIa binding inhibitory activities of 2,3-dihydro-1,4- benzodioxine derivatives through regio- and stereoisomerism

Article abstract of DOI:10.1016/j.ejmech.2013.01.002

Enantiomers of 2,3-dihydro-1,4-benzodioxine derivatives possessing both thrombin and fibrinogen GPIIb/IIIa binding inhibitory activities were prepared from (R)- and (S)-glycidol as potential dual antithrombotic compounds. The influence of chirality and su

Full text of DOI:10.1016/j.ejmech.2013.01.002

European Journal of Medicinal Chemistry 62 (2013) 329e340
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Towards dual antithrombotic compounds e Balancing thrombin
inhibitory and fibrinogen GPIIb/IIIa binding inhibitory activities
of 2,3-dihydro-1,4-benzodioxine derivatives through regio- and
stereoisomerism
b
c
c
ꢀ
ꢁ a
ꢀa
Milos Ilic , Petra Dunkel , Janez Ilas , Ewa Chabielska , Agnieszka Zakrzeska ,
**
Péter Mátyus b , Danijel Kikelj
,
a,
*
a
ꢀ
ꢀ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia
b Semmelweis University, Faculty of Pharmacy, Department of Organic Chemistry, Högyes E. u. 7, 1092 Budapest, Hungary
c Medical University of Bialystok, Department of Biopharmacy, Mickiewicza 2c, 15-089 Bialystok, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Enantiomers of 2,3-dihydro-1,4-benzodioxine derivatives possessing both thrombin and fibrinogen
GPIIb/IIIa binding inhibitory activities were prepared from (R)- and (S)-glycidol as potential dual
antithrombotic compounds. The influence of chirality and substitution pattern on thrombin inhibition
and on inhibition of fibrinogen binding to GPIIb/IIIa was analyzed. Docking studies were used in an
attempt to rationalize the results. The (S)-isomers of both 2,3-dihydro-1,4-benzodioxine regioisomers at
positions 6 and 7 were found to be better thrombin inhibitors than the corresponding (R)-enantiomers,
whereas we observed that stereochemistry does not display a consistent influence on fibrinogen GPIIb/
IIIa binding inhibitory activity. Compound 11b, the (S)-isomer of the 6-substituted regioisomer, pos-
Received 8 November 2012
Received in revised form
22 December 2012
Accepted 3 January 2013
Available online 10 January 2013
Keywords:
Antithrombotic
sessed the best balanced dual activity, with Ki(thrombin)
¼ 0.665 ꢀ 0.26 M, raising the hope that merging anticoagulant and platelet antiaggregatory ac-
tivities in the same molecule could lead to successful multitarget antithrombotic agents.
Ó 2013 Elsevier Masson SAS. All rights reserved.
¼
1.67
ꢀ
0.27 mM and IC50(GPIIb/
Thrombin inhibitor
GPIIb/IIIa
2,3-dihydro-1,4-benzodioxine
Dual antithrombotic compound
Multitarget
m
IIIa)
1. Introduction
for the existing warfarin treatment [4e11]. Ximelagatran, a prodrug
of melagatran, was the first oral direct thrombin inhibitor intro-
duced to the market, however it was withdrawn from the market in
2006 due to expressed hepatotoxicity [12,13]. Dabigatran etexilate
is a new oral prodrug for the direct thrombin inhibitor dabigatran
(Fig. 1) that entered the market in 2008 for the prevention of blood
clotting following hip or knee surgery, and for the prevention of
stroke in patients with non-valvular atrial fibrillation [14]. Good
progress has also been made in the field of direct and indirect factor
Xa inhibitors. Rivaroxaban, a recently introduced, highly selective
oral factor Xa inhibitor, promised effectiveness and safety in pre-
venting venous thromboembolism [15]. Other novel factor Xa in-
hibitors include the recently approved apixaban, and idraparinux
that is in the final stage of clinical investigation [16].
Cardiovascular diseases are the main cause of death in devel-
oped countries [1]. Existing anticoagulant and antiplatelet therapy,
despite its importance and effectiveness in the treatment of car-
diovascular diseases, has numerous limitations such as bleeding,
metabolism that differs in different individuals due to genetic
polymorphism, and interaction with other drugs and food [2,3].
During the last decade much effort have been made to design
novel anticoagulant drugs that target the coagulation enzymes
thrombin, factor Xa, factor VII and factor IX, with the aim of
obtaining a simple and satisfactory, orally applicable replacement
Abbreviations: GPIIb/IIIa, glycoprotein IIb/IIIa; PDB, Protein Data Bank; DMAP,
dimethylaminopyridine; DCM, dichloromethane; DMF, N,N-dimethylformamide;
DIAD, diisopropylazodicarboxylate; TFA, trifluoroacetic acid.
* Corresponding author. Tel.: þ386 1 4769561; fax: þ386 1 4258031.
** Corresponding author.
Platelets play an essential role in the maintenance of hemosta-
sis; they are responsible for clot formation when damage of
endothelium of blood vessels occurs [17]. The platelet glycoprotein
IIb/IIIa antagonists abciximab, eptifibatide and tirofiban are potent
antiplatelet agents, and are used to prevent pathological platelet
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.2013.01.002

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M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
330
substituents in positions 2, 4, 6 and 7 [25]. The benzamidine moiety,
CH3
HN
NH2
NH
a typical P1 group of thrombin inhibitors and an arginine mimetic
of the RGD motif [28], was attached at position 2 of the benzoxazine
core, while P3 benzyl and carboxylic acid moieties, required for
thrombin inhibition and GPIIb/IIIa binding [29], were bound at
positions 6 or 7 of the 1,4-benzoxazine skeleton (Fig. 1). Molecular
modeling studies predicted 7-isomers to be better thrombin in-
hibitors while 6-isomers were expected to possess better inhibition
of fibrinogen GPIIb/IIIa binding, as was later confirmed in biological
assays [25]. Focusing on optimization of the P3 part, with a com-
bination of different aromatic and carboxylate group moieties, we
succeeded in preparing well-balanced dual compounds with
thrombin Ki values and IC50 values for inhibition of fibrinogen
binding to platelet GPIIb/IIIa in the high nanomolar/low micro-
molar ranges [30]. Replacement of the highly basic benzamidine
moiety with the less basic [1,2,4] triazolo[4,3-b]pyridazine-6-yl
group resulted in loss of both thrombin inhibitory and fibrinogen
receptor antagonistic activities [31], showing the importance of the
benzamidine group for binding to both targets. The failure to ach-
ieve better affinities of the designed compounds for thrombin and
fibrinogen receptor demonstrates the difficulty in reaching a com-
promise that would meet the constraints imposed by both binding
sites.
N
O
N
HO
N
N
O
dabigatran
NH2
NH
X
X
N
N
CH3
O
N
O
O
R:
R
O
O
HO
HO
CH3
X = H, 3'-F, 4'-F, 3',4'-diF, 3',5'-diF,
previously reported racemic 6- and 7-substituted 1,4-benzoxazine derivatives
NH2
NH
∗
O
O
N
R = H, Et
O
O
O
RO
When designing multiple ligands with highly overlapping
pharmacophores, balancing activities for both targets is demand-
ing, so all structural information is of the utmost value [32e35]. In
the present work we report a 1,4-benzodioxine series of enantio-
meric compounds that combine, in the same molecule, highly
overlapping pharmacophores of thrombin inhibitors and fibri-
nogen receptor antagonists (Fig.1). They resulted from replacement
of the 1,4-benzoxazine core by a 1,4-benzodioxine scaffold [36],
a privileged heterocyclic skeleton applied in the design of selective
a1 adrenoceptor antagonists [37], antioxidants [38,39], radical
scavenging compounds [40e42], hypolipidemic [43] and anti-
inflammatory agents [44,45]. In order to explore the significance
of chirality and regioisomerism in balancing the activity of the new
1,4-benzodioxine compounds at both targets, and thanks to the
commercial availability of both (R)- and (S)-glycidol (required for
enantioselective synthesis of the parent 2-substituted benzodiox-
ine cores), we prepared both enantiomers of the benzodioxine 6-
and 7-regioisomers and studied the effects of chirality and regioi-
somerism on binding the resulting potential dual antithrombotic
compounds to thrombin and platelet fibrinogen receptor.
new enantiomers of 6- and 7-substituted 1,4-benzodioxines
Fig. 1. Structures of the clinically used thrombin inhibitor dabigatran, the racemic 1,4-
benzoxazine derivatives [25,30] and the enantiomers of 1,4-benzodioxine derivatives
reported in this paper.
aggregation in patients with acute coronary syndrome, heart attack
and those undergoing invasive heart procedures [18].
In clinical practice, an effective prophylaxis and treatment of
thromboembolic diseases, such as deep vein thrombosis, throm-
boembolic stroke, pulmonary embolism and myocardial infarction,
is achieved by using a combination of various antiaggregatory and
anticoagulant drugs [19]. Since the combined use of thrombin in-
hibitors and glycoprotein IIb/IIIa antagonists in the prevention of
cardiovascular diseases has shown additional benefits over treat-
ment directed against thrombin or against platelets alone [20], we
envisaged merging anticoagulant and platelet antiaggregatory ac-
tivity in the same molecule as a promising approach towards novel
multitarget antithrombotic agents [21e25]. Recently, other groups
have reported the preparation of dual anticoagulant/antiplatelet
agents featuring high molecular weight, polysulfated conjugates
[26], in contrast to our dual acting compounds with strongly
overlapping pharmacophores and low molecular weight.
2. Chemistry
In our previous work on potential dual antithrombotic com-
pounds, combining in one molecule both thrombin inhibitory and
fibrinogen receptor antagonistic activities [24,25], 3,4-dihydro-2H-
1,4-benzoxazine [27] was selected as a suitable scaffold, on the
basis of docking experiments, enabling convenient attachment of
The synthesis of target 1,4-benzodioxine enantiomers 11ae
d from 4-nitrocatechol is presented in Schemes 1 and 2. The
strongly electronegative nitro group in 4-nitrocatechol enabled
effective control of the regioselectivity in construction of the 1,4-
Reagents and conditions: a) Et N, DMAP, DCM, 0 ºC → rt, 1h; b) NaH, DMF, 80 ºC, 2h; c)
Na2CO3, DMF, 60 ºC, 2h.
3
Scheme 1. Synthesis of compounds 5aed.

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M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
331
Reagents and conditions: a) 4-cyanophenol, PPh3, DIAD, THF, reflux, 48h; b) H2, Pd/C
(10%), 25 bar, rt, 1h; c) benzaldehyde, MeOH, mol. sieves, rt, 12h, then NaBH4, 1h; d)
EtOCOCOCl, Et3N, DCM, rt, 2h; e) HCl(g), EtOH, 0 ºC (30 min) → rt, 24h, then
CH3COONH4, 24h; f) 1M LiOH, THF/MeOH, rt, 1h.
Scheme 2. Synthesis of compounds 10aed and 11aed.
benzodioxine ring and, depending on the applied base, gave rise to
6- and 7-nitro isomers, respectively [46]. Thus, using a modified
published procedure for the synthesis of the racemates [47], the
reaction of 4-nitrocatechol with (R)- and (S)-(2-tosyloxymethyl)
oxirane (3b and 3a), in the presence of potassium carbonate in
N,N-dimethylformamide afforded enantiomers of (7-nitro-2,3-
dihydro-1,4-benzodioxin-2-yl)methanol (5d and 5c), whereas
the reaction of 4-nitrocatechol with 3a or 3b, using sodium hy-
dride as a base, gave (R)- and (S)-6-nitro isomers 5a and 5b
(Scheme 1). Both nitro isomers of the (R)- and (S)-series were
reacted with 4-hydroxybenzonitrile under Mitsunobu conditions
to give ethers 6aed, which were reduced in the next step to
amines 7aed, using catalytic hydrogenation over palladium on
charcoal. These were benzylated, using benzaldehyde and sodium
borohydride, and the resulting N-benzylamines 8aed acylated
with ethyl oxalyl chloride to give compounds 9aed. Upon Pinner
reaction they afforded esters 10aed which, after alkaline hydro-
lysis with lithium hydroxide in methanol/tetrahydrofuran, gave
the enantiomeric carboxylic acids 11aed (Scheme 2). We tried to
develop an HPLC method for determining the enantiomeric purity
of products 5e11, but succeeded only with compounds 8aed for
which an enantiomeric excess >95% was obtained. The non-
racemizing conditions in the last two reaction steps and con-
sistent optical rotation values of the products allow the conclusion
that the enantiomeric excess of compounds 10aed and 11ae
d remained higher than 95%.
Table 1
Biological activity of 1,4-benzodioxine derivatives 10aed and 11aed: the inhibition of serine proteases thrombin, factor Xa, trypsin, and the inhibition of fibrinogen binding to
GPIIb/IIIa.
Compound
Substitution
R0
*
Ki (
m
M)
IC50 (mM)
Thrombin
Trypsin [selectivity]a
FXa [selectivity]b
GPIIb/IIIac
10a
10b
10c
10d
11a
11b
11c
11d
6
6
7
7
6
6
7
7
Et
Et
Et
Et
H
H
H
H
R
S
R
S
R
S
R
S
1.65 ꢀ 0.20
0.644 ꢀ 0.089 [0.39]
0.283 ꢀ 0.044 [0.65]
0.894 ꢀ 0.097 [1.32]
0.210 ꢀ 0.026 [2.71]
1.55 ꢀ 0.22 [0.30]
2.15 ꢀ 0.18 [1.30]
1.51 ꢀ 0.37 [3.46]
0.637 ꢀ 0.44 [0.95]
2.70 ꢀ 0.15 [35.9]
5.19 ꢀ 0.61 [1.30]
1.96 ꢀ 0.17 [1.17]
1.27 ꢀ 0.13 [0.47]
5.08 ꢀ 0.57 [19.1]
>200
>200
>200
>200
1.21 ꢀ 0.32
0.665 ꢀ 0.26
17.6 ꢀ 3.0
42.6 ꢀ 5.3
0.436 ꢀ 0.037
0.675 ꢀ 0.099
0.0778 ꢀ 0.005
5.18 ꢀ 1.15
1.67 ꢀ 0.27
0.463 ꢀ 0.093 [0.28]
1.37 ꢀ 0.029 [0.51]
0.267 ꢀ 0.049 [1.00]
2.69 ꢀ 0.36
0.266 ꢀ 0.03
a
Selectivity for thrombin vs. trypsin.
Selectivity for thrombin vs. factor Xa.
b
c
Compounds were also tested for inhibition of fibrinogen binding to aVb3 receptor and none of them showed binding in the test system (IC50(aVb3) > 200 mM).

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M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
332
3. Biological results
thrombin inhibitory activity (Ki(thr) ¼ 0.19 ꢀ 0.02
mM, IC50(GPIIb/
¼ 39 ꢀ 18 M) [26]. When considering eudismic ratios, the
m
IIIa)
Compounds 10aed and 11aed, comprising enantiomers of the
6- and 7-isomers, were tested for inhibition of thrombin and, in
order to assess their selectivity for related serine proteases, also for
inhibition of factor Xa and trypsin. Additionally, they were tested
for inhibition of fibrinogen binding to GPIIb/IIIa. The results are
presented in Table 1. The general structureeactivity relationship
which was observed in the 1,4-benzoxazine series can also be seen
in the case of the 1,4-benzodioxine series. Esters 10aed were found
to be more potent thrombin inhibitors than the corresponding
carboxylic acids 11aed, but they lacked fibrinogen GPIIb/IIIa
results in Table 1 demonstrate that, in the case of esters 10aed in
the 6-regioisomer series, the (S)-isomer is 3.8 times more potent
and, in the 7-regioisomer series, 8.7 times more potent thrombin
inhibitor than corresponding (R)-isomer. Similar results are seen in
the case of acids 10aed where, in 6-substituted compounds, the
(S)-isomer is 3.1 times more potent and, in the 7-substituted
compound, 10.1-fold more potent as a thrombin inhibitor. Stereo-
chemistry does not appear to have such a consistent influence on
fibrinogen GPIIb/IIIa binding inhibitory activity. In the case of the 6-
substituted compound, the (S)-isomer 11b is 1.8 times more potent
than the (R)-isomer 11a, while in case of 7-substituted compounds
the (R)-isomer 11c is 2.4 times more potent than the (S)-isomer 11d.
It can thus be concluded that, in the 1,4-benzodioxine series, the
(S)-6-isomers offer the best opportunities for optimization towards
balanced dual acting compounds.
binding activity (IC50 > 200
in position 6 (11aeb) displayed more pronounced fibrinogen GPIIb/
IIIa binding inhibitory activity (IC50 0.66 and 1.21 M), while
compounds bearing a P3 moiety at position 7 exhibited more
M).
mM). Compounds bearing a P3 moiety
m
pronounced thrombin inhibitory activity (Ki ¼ 0.078e2.69
m
While compounds 10aed and 11aed were not selective towards
two similar serine proteases, trypsin and factor Xa, the compound
with the most potent thrombin inhibitory activity (10d) was found
also to be the most selective. As demonstrated in Fig. 1, compound
11b, the (S)-isomer of the 6-substituted regioisomer, possessed the
4. Docking studies
In an attempt to rationalize the differences in the binding af-
finities of (R)- and (S)-enantiomers for thrombin and fibrinogen
receptor, we performed docking of compounds 11a and 11b ‒ both
enantiomers of the more balanced 6-regioisomer docked to the
thrombin active site and to the binding site of GPIIb/IIIa are
depicted in Fig. 2. Given the similarity of our compounds to dabi-
gatran, the crystal structure of the dabigatranethrombin complex
(PDB: 1KTS) [48] was used for docking experiments. Compounds
11a and 11b bind to the thrombin active site in a similar manner to
dabigatran but the configuration at chiral centers slightly in-
fluences the difference in their binding modes. The distance be-
tween the C-atoms of the Asp189 carboxylate and the amidine
group is 3.9 Å and 4.0 Å for 11a and 11b, and two hydrogen bonds
connect the amidine hydrogens and carboxylate oxygen atoms.
Additional hydrogen bonds of 1.98 Å and 2.0 Å between the ben-
zamidine and the Gly219 backbone for the two compounds are
observed. The 2,3-dihydro-1,4-benzodioxine scaffold is located in
the S2 binding pocket and the benzyl group reaches into the lip-
ophilic S3 binding pocket, with the P3 carboxylate stretching
best balanced dual activity (Ki(thr) ¼ 1.67 ꢀ 0.27
mM, IC50(GPIIb/
¼ 0.665 ꢀ 0.26 M). Compound 10d, the (S)-isomer of the
m
IIIa)
7-regioisomer, is the most potent thrombin inhibitor, with
Ki(thr) ¼ 77.8 ꢀ 5.0 nM, and compound 11b, ((S)-isomer of the 6-
regioisomer) has the most potent fibrinogen GPIIb/IIIa binding
inhibitory activity, with IC50(IIb/IIIa) ¼ 665 ꢀ 260 nM. This demon-
strates that, in the development of dual antithrombotic compounds
based on the 1,4-benzodioxine scaffold, optimizing fibrinogen re-
ceptor activity is a more difficult task than optimizing thrombin
inhibition.
The (S)-isomers of the 6- and 7-series were found to be better
thrombin inhibitors than the (R)-isomers. Overall, these results are
comparable with those for the 1,4-benzoxazine analogues. The
racemic 1,4-benzoxazine derivative substituted at position
showed higher activity on both targets (Ki(thr) ¼ 0.62 ꢀ 0.10
IC50(GPIIb/IIIa) ¼ 2.8 ꢀ 1.5
derivative substituted at position
6
M,
m
m
M), while the racemic 1,4-benzoxazine
7
had more pronounced
Fig. 2. Compounds 11a (R-isomer, left) and 11b (S-isomer, right) docked to thrombin active site (top) and GPIIb/IIIa binding site (bottom).

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M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
333
outwards from the thrombin surface. The methyleneoxy groups
6. Experimental section
adjacent to the chiral carbon of 3,4-dihydro-1,4-benzodioxine are
oriented differently in the two enantiomers. The distance between
the oxygen of the methyleneoxy group and the Ser195 OH group
(OeHꢁꢁꢁO) is 3.1 Å in 11b and 3.6 Å in 11a. The carbon atom of the
methylenoxy groups adjacent to the chiral center is the atom that
differs most in its positioning in 11a and 11b ‒ by 1.92 Å. In 11b, the
methylene group is closer to the protein surface while, in 11a, it
points outwards from the thrombin surface.
6.1. General
Chemicals were obtained from Acros, Aldrich Chemical Co. and
Fluka and used without further purification. Analytical TLC was
performed on silica gel Merck 60 F254 plates (0.25 mm), using
visualization with ultraviolet light. Column chromatography was
carried out on silica gel 60 (particle size 240e400 mesh). Melting
points were determined on a Reichert hot stage microscope and are
uncorrected. 1H NMR and 13C NMR spectra were recorded on
a 400 MHz Bruker AVANCE III NMR spectrometer (1H NMR at
400.13 MHz and 13C NMR at 100.61 MHz) and on a Bruker Avance
DPX300 spectrometer (1H NMR at 300 MHz, 13C NMR at 75 MHz) in
DMSO-d6 solution, with TMS as internal standard. 13C NMR spectra
were assigned using gradient COSY, HSQC and HMBC experiments.
The following abbreviations were used to describe peak splitting
The analysis of the binding modes of 11a and 11b docked in the
binding site of GPIIb/IIIa (using PDB: 2VDM ‒ the only published
crystal structure of GPIIb/IIIa in a complex with a low molecular
weight RGD mimetic) [49] shows that the compounds take an
extended conformation. The carboxylic acid moiety occupies an
identical location in 11a and 11b, making electrostatic interactions
with the Mg2þ atom and hydrogen-bonding to the amide proton of
Asn215 of the b3 subunit of the GPIIb/IIIa. The phenyl rings of 11a
and 11b also occupy identical positions, making hydrophobic in-
teractions with Tyr122 of the b3 subunit. The methylenoxy groups
and the benzamidine moieties of 11a and 11b occupy slightly dif-
ferent positions. The Ala218 of the b3 subunit makes, with its
methyl group, hydrophobic interactions with the exocyclic meth-
ylenoxy group of the ligand (distances of 4.2 Å for 11b and 5.5 Å for
11a) and polar interactions of its C]O group with the exocyclic
ether oxygen of the ligand (2.9 Å for 11b and 3.3 Å for 11a). Further,
positioning of the benzamidine moiety, which forms hydrogen
bonds with Asp224 and the Phe160 from the aIIb subunit of the
GPIIb/IIIa, and hydrophobic interactions with Tyr190, Phe231, and
Phe191 of the aIIb subunit, differs between the R- and S-isomers by
0.94 Å difference in the position of the carbon atom of the amidine
moieties of 11a and 11b. The docking experiments thus show better
binding interactions of 11b than that of 11a to platelet fibrinogen
receptor (GPIIb/IIIa).
patterns wherever appropriate: br
¼
broad,
d
¼
doublet,
dd ¼ doublet of doublet, t ¼ triplet, q ¼ quartet, and m ¼ multiplet.
IR spectra were recorded on a PerkineElmer 1600 FT-IR spec-
trometer. Microanalyses were performed on a PerkineElmer C, H, N
Analyzer 240 C. Analyses indicated by the symbols of the elements
were within ꢀ0.4% of the theoretical values. Mass spectra were
obtained using a VG-Analytical Autospec Q mass spectrometer.
Optical rotations were measured on a PerkineElmer 1241 MC
polarimeter. Reverse phase HPLC was performed on an Agilent
Technologies HP 1100 instrument with G1365B UVevis detector
(254 nm), using an Eclipse Plus C18 column 5
mm (4.6 ꢂ 150 mm)
at flow rate of 1 mL/min. The eluent was a mixture of 0.1% TFA in
water (A) and methanol (B) and the gradient was 40% B to 80% B
in 15 min. Chiral HPLC analyses were performed on a JASCO
ChromPass Software Version 1.8 with JASCO UV-2075 UVevis de-
tector (244 nm), using a Chiralcel OJ-H 5
m
m 4.6 ꢂ 250 mm column
In summary, the measured activities and analysis of the docking
positions of 11a and 11b at the two targets lead to the conclusion
that the configuration at the chiral center, and the consequent
different positioning of the methylenoxy moiety, accounts, at least
partly, for the difference in potency of the two enantiomers.
Whereas the methylenoxy moiety situated in close proximity to the
chiral center is required for flexibility of the molecule (compounds
10ae11d adopt a bent shape in the thrombin active site and
extended conformations in the fibrinogen receptor-binding site),
this could also explain the low eudismic ratios resulting from the e
CH2Oe group imposed molecular flexibility that diminishes the
difference between the binding conformations of two enantiomers
at a particular target.
at flow rate of 1 mL/min. Compounds were separated under iso-
cratic conditions using as eluent a mixture of 60% n-hexane and 40%
ethanol. All the compounds reported in this paper were >95% pure.
Esters 10aed were purified by reverse phase column chromatog-
raphy, using a Flash Purification System ISOLERAÔ. The eluent was
a mixture of 0.1% TFA in water (A) and methanol (B) and the gra-
dient was 40% B to 80% B in 30 column volumes.
6.2. Synthesis of (R)-glycidyl tosylate (3b)
To a solution of (S)-glycidol (5.50 g, 74.24 mmol), triethylamine
(8.26 g, 81.66 mmol) and 4-(dimethylamino)pyridine (2%, 181 mg,
1.48 mmol) in 60 mL dichloromethane at 0 ꢃC, tosyl chloride
(14.15 g, 74.24 mmol) was added and the mixture was stirred at
room temperature for 3 h with formation of a white precipitate.
Dichloromethane (40 mL) was added and the mixture was subse-
quently washed with 100 mL 5% aq. K2CO3, 100 mL 1 M HCl, and
100 mL water. The organic layer was dried over Na2SO4, filtered and
evaporated to dryness. The crude product (15.56 g colourless oil,
92% yield) was used in the next step without further purification;
5. Conclusion
A novel 1,4-benzodioxine enantiomeric series of compounds
have been prepared that act both as thrombin inhibitors and as
fibrinogen GPIIb/IIIa binding inhibitors. The influence of chirality
and regioisomerism on their thrombin inhibitory and fibrinogen
GPIIb/IIIa binding inhibitory activities has been analyzed. The dif-
ferences in potency of isomers and observed eudismic ratios can be
explained in terms of the docking poses of the two stereoisomers.
Unfortunately, we were not able to observe antithrombotic action
of this series of compounds in in vivo model of venous thrombosis
in rat, indicating that their potencies on both targets are still too
weak to be measured in vivo. The attempt to balance thrombin
inhibition and fibrinogen GPIIb/IIIa binding inhibitory activities on
the one hand and to achieve lower nanomolar Ki (thrombin) and
IC50 (GPIIb/IIIa) values on the other ‒ which could result in in vivo
antithrombotic activity ‒ remains a viable, but highly demanding
challenge in the search for effective dual antithrombotic drugs.
[a
]
D
¼ ꢄ16.7 (c ¼ 0.46, CHCl3), lit.: [50] [
a
]
¼ ꢄ17.0 (c ¼ 2.83,
D
CHCl3); 1H NMR (300 MHz, CDCl3):
d (ppm) 2.47 (s, 3H, CH3), 2.61
(dd, J ¼ 4.8, 2.5 Hz,1H, CH2O), 2.83 (t, J ¼ .4 Hz,1H, CH2O), 3.16e3.24
(m, 1H, CH), 4.0 (dd, J ¼ 11.4, 6.0 Hz, 1H, CH2OS), 4.27 (dd, J ¼ 11.4,
3.5 Hz, 1H, CH2OS), 7.37 (d, J ¼ 8.1 Hz, 2H, AreH3, AreH5), 7.82 (d,
J ¼ 8.1 Hz, 2H, AreH2, AreH6).
6.3. Synthesis of (S)-glycidyl tosylate (3a)
To a solution of (R)-glycidol (4.00 g, 54.00 mmol), triethylamine
(6.01 g, 59.50 mmol) and 4-(dimethylamino)pyridine (2%, 132 mg,
1.08 mmol) in 50 mL dichloromethane at 0 ꢃC, tosyl chloride (10.30 g,

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334
54.00 mmol) was added and the mixture was stirred at room tem-
perature for 1 h. During stirring a white precipitate was formed. The
reaction mixture was subsequently washed with 100 mL 5% aq.
K2CO3, 100 mL 1 M HCl, and 100 mL water. The organic layer was
dried over Na2SO4, filtered and evaporated to dryness. The crude
product (11.63 g colourless oil, 95% yield) was used in the next step
[52] þ65.5 (c ¼ 0.58, 96% EtOH); 1H NMR (300 MHz, CDCl3):
d
(ppm) 1.94 (t, J ¼ 6.21 Hz, 1H, OH), 3.84e4.08 (m, 2H, CH2OH),
4.12e4.28 (m, 1H, CH), 4.28e4.49 (m, 2H, CH2O), 6.94e7.02 (m, 1H,
AreH5), 7.74e7.87 (m, 2H, AreH6, AreH8).
6.7. Synthesis of (S)-(7-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)
without further purification; [
a
]
¼ þ16.3 (c ¼ 0.57, CHCl3), lit.: [51]
methanol (5d)
D
[a]
¼ þ17.5 (c ¼ 2.13, CHCl3); 1H NMR (300 MHz, CDCl3):
d (ppm)
D
2.47 (s, 3H, CH3), 2.61 (dd, J ¼ 4.8, 2.5 Hz,1H, CH2O), 2.81e2.85 (m,1H,
CH2O), 3.17e3.24 (m, 1H, CH), 3.98 (dd, J ¼ 11.4, 6.0 Hz, 1H, CH2OS),
4.27 (dd, J ¼ 11.4, 3.5 Hz, 1H, CH2OS), 7.37 (d, J ¼ 8.1 Hz, 2H, AreH3,
AreH5), 7.83 (d, J ¼ 8.1 Hz, 2H, AreH2, AreH6).
A suspension of 4-nitrocatechol (310 mg, 2.00 mmol), (R)-gly-
cidyl tosylate (545 mg, 2.40 mmol) and K2CO3 (663 mg, 4.80 mmol)
in 5 mL DMF was heated at 60 ꢃC for 2 h. The mixture was poured
onto 15 mL water and extracted with 4 ꢂ 20 mL diethyl ether. The
combined organic phases were subsequently washed with
3 ꢂ 20 mL 10% aq. K2CO3, 20 mL water and 20 mL brine. The organic
layer was dried over Na2SO4, filtered and evaporated to dryness.
The crude product obtained was purified by column cromatography
(petrolether:ethyl acetate ¼ 1:1) to give 180 mg of yellow crystals
6.4. Synthesis of (R)-(6-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)
methanol (5a)
To a suspension of NaH (60% dispersion, 120 mg, 1.5 mmol) in
5 mL DMF at 0 ꢃC, 4-nitrocatechol (310 mg, 2.00 mmol) in 1 mL DMF
was added carefully. The mixture was stirred at 0 ꢃC for 30 min
(until gas formation ceased) and then a solution of (S)-glycidyl
tosylate (502 mg, 2.20 mmol) in 1 mL DMF was added. The reaction
mixture was stirred at room temperature for 30 min and then
heated at 80 ꢃC for 2 h. Upon cooling, the mixture was poured onto
15 mL water. The mixture was extracted with 3 ꢂ 20 mL diethyl
ether. The combined organic phases were dried over Na2SO4, fil-
tered and evaporated to dryness. The crude product obtained was
purified by column cromatography (petrolether:ethyl acetate 1:1)
to give 178 mg (42%) of pale yellow crystals; mp 112e114 ꢃC;
(43%); mp 117e119 ꢃC; [
(300 MHz, CDCl3):
a
]
¼ ꢄ61.6 (c ¼ 0.5 in CHCl3); 1H NMR
D
d
(ppm) 1.93 (t, J ¼ 6.19 Hz, 1H, OH), 3.86e4.04
(m, 2H, CH2OH), 4.13e4.27 (m, 1H, CH), 4.30e4.47 (m, 2H, CH2O),
6.93e7.03 (m, 1H, AreH5), 7.75e7.86 (m, 2H, AreH6, AreH8).
6.8. (R)-4-((6-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)methoxy)
benzonitrile (6a). General procedure for synthesis of compounds
6aed
The benzodioxine alcohol 5a (2.80 g, 13.26 mmol) was dissolved
in anhydrous THF (50 mL). Under argon flow and with ice/water
cooling, 4-cyanophenol (1.74 g, 14.59 mmol) and triphenylphos-
phine (6.96 g, 26.52 mmol) were added. Diizopropyl azodicarbox-
ylate (DIAD) (5.36 g, 26.52 mmol) dissolved in 15 mL anhydrous
THF was added dropwise at 0 ꢃC. The solution was stirred for
30 min at 0 ꢃC, and then heated to reflux for 48 h. The mixture was
evaporated to dryness and the crude product was purified by
recrystallization from MeOH to give 2.15 g (52%) of 6a as pale yel-
[
d
a
]
¼ þ87.61 (c ¼ 0.57, CHCl3); 1H NMR (300 MHz, CDCl3):
D
(ppm) 1.91 (t, J ¼ 6.20 Hz, 1H, OH), 3.86e4.04 (m, 2H, CH2OH),
4.09e4.28 (m, 1H, CH), 4.33e4.47 (m, 2H, CH2O), 6.94e7.08 (m, 1H,
AreH8), 7.75e7.90 (m, 2H, AreH5, AreH7).
6.5. Synthesis of (S)-(6-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)
methanol (5b)
low crystals; mp 159e162 ꢃC; [
NMR (400 MHz, DMSO-d6):
a]
¼ þ83.4 (c ¼ 0.5 in CDCl3); 1H
D
To a suspension ofNaH (60% dispersion,120 mg,1.5mmol) in 5 mL
DMF at 0 ꢃC, 4-nitrocatechol (310 mg, 2.00 mmol) in 1 mL DMF was
added. The mixture was stirred at 0 ꢃC for 30 min (until gas formation
ceased) and then a solution of (R)-glycidyl tosylate (502 mg,
2.20 mmol) in 1 mL DMF was added. The reaction mixturewas stirred
at room temperature for 30 min and then heated at 80 ꢃC for 2 h.
Upon cooling, themixturewas poured onto 15 mL water. Themixture
was extracted with 3 ꢂ 20 mL diethyl ether. The combined organic
phases were dried over Na2SO4, filtered and evaporated to dryness.
The crude product obtained was purified by column cromatography
(petrolether:ethyl acetate ¼ 1:1) to give 212 mg (50%) of pale yellow
d
(ppm) 4.28 (dd, J ¼ 11.8, 7.2 Hz, 1H, 3-
CH2), 4.39 (dd, J ¼ 11.1, 5.7 Hz, 1H, CH2O), 4.46 (dd, J ¼ 11.1, 3.7 Hz,
1H, CH2O), 4.58 (dd, J ¼ 11.8, 2.5 Hz, 1H, 3-CH2), 4.77e4.85 (m, 1H,
2-CH), 7.13e7.22 (m, 3H, AreH8, AreH2’, AreH6’), 7.77e7.84 (m, 4H,
AreH5, AreH7, AreH3’, AreH5’); 13C NMR (101 MHz, DMSO-d6):
d
(ppm) 64.4 (C-3), 66.5 (CH2O), 72.2 (C-2), 103.4 (C-40), 112.7 (C-7),
115.6 (C-20, C-60), 117.6, 117.7 (C-5, C-8), 119.0 (CN), 134.2 (C-30, C-50),
141.1 (C-6), 142.7 (C-4a), 148.8 (C-8a), 161.4 (C-10); HRMS (ESI) m/z
calcd for C16H13N2O5 [M þ H]þ 313.0824, found 313.0830; IR (KBr, v,
cmꢄ1): 2222, 1605, 1522, 1471, 1347, 1252, 1174, 839; HPLC: 100%, tr
16.0 min; Anal. (C16H12N2O5) C, H, N.
crystals; mp 112e114 ꢃC; [
(300 MHz, CDCl3):
2H, CH2OH), 4.10e4.25 (m, 1H, CH), 4.33e4.46 (m, 2H, CH2O), 6.95e
7.04 (m, 1H, AreH8), 7.76e7.88 (m, 2H, AreH5, AreH7).
a
]
¼ ꢄ86.6 (c ¼ 0.51, CHCl3); 1H NMR
D
d
(ppm) 1.91 (t, J ¼ 6.20 Hz,1H, OH), 3.85e4.04 (m,
6.9. (S)-4-((6-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)methoxy)
benzonitrile (6b)
Synthesized from 5b (1.17 g, 5.54 mmol) according to the gen-
eral procedure for synthesis of compounds 6aed; pale yellow
6.6. Synthesis of (R)-(7-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)
methanol (5c)
crystals, yield 865 mg (50%); mp 172e175 ꢃC; [
a
]
¼ ꢄ80.4 (c ¼ 0.5,
D
CDCl3); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 4.33 (dd, J ¼ 11.7,
A suspension of 4-nitrocatechol (310 mg, 2.00 mmol), (S)-gly-
cidyl tosylate (545 mg, 2.40 mmol) and K2CO3 (663 mg, 4.80 mmol)
in 5 mL DMF was heated at 60 ꢃC for 2 h. The mixture was poured
onto 15 mL water and extracted with 3 ꢂ 20 mL diethyl ether. The
combined organic phases were subsequently washed with
3 ꢂ 20 mL 10% aq. K2CO3, 20 mL water and 20 mL brine. The organic
layer was dried over Na2SO4, filtered and evaporated to dryness.
The crude product obtained was purified by column cromatography
(petrolether:ethyl acetate ¼ 1:1) to give 191 mg of pale yellow
7.3 Hz, 1H, 3-CH2), 4.38 (dd, J ¼ 11.1, 5.6 Hz, 1H, CH2O), 4.45 (dd,
J ¼ 11.1, 3.7 Hz, 1H, CH2O), 4.62 (dd, J ¼ 11.7, 2.5 Hz, 1H, 3-CH2),
4.72e4.78 (m, 1H, 2-CH), 7.11e7.23 (m, 3H, AreH8, AreH2’, AreH6’),
7.76e7.84 (m, 4H, AreH5, AreH7, AreH3’, AreH5’); 13C NMR
(101 MHz, DMSO-d6):
d (ppm) 65.0 (C-3), 66.5 (CH2O), 71.4 (C-2),
103.4 (C-40), 112.7 (C-7), 115.7 (C-20, C-60), 117.5, 117.6 (C-5, C-8),
119.0 (CN), 134.2 (C-30, C-50), 141.2 (C-6), 142.4, 149.0 (C-4a, C-8a),
161.4 (C-10); HRMS (ESI) m/z calcd for C16H13N2O5 [M þ H]þ
313.0824, found 313.0813; IR (KBr, v, cmꢄ1): 2227, 1603, 1514, 1349,
1256, 1174, 839; HPLC: 100%, tr 16.0 min; Anal. (C16H12N2O5) C, H, N.
crystals (45%); mp 117e119 ꢃC; [
a]
D
¼ þ59.72 (c ¼ 0.5 in CHCl3), lit.:

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335
6.10. (R)-4-((7-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)methoxy)
benzonitrile (6c)
7.25 (m, 1H, Ph), 7.27e7.39 (m, 4H, Ph), 7.78 (d, J ¼ 9.0 Hz, 2H, Are
H3’, AreH5’); 13C NMR (101 MHz, DMSO-d6):
(ppm) 47.0 (PheCH2),
d
64.7 (C-3), 66.9 (CH2O), 70.7 (C-2), 100.4 (C-5), 103.2 (C-40), 106.5
(C-7), 115.6 (C-20, C-60), 117.2 (C-8), 119.0 (CN), 126.5 (C-400), 127.1 (C-
200, C-600), 128.2 (C-300, C-500), 133.3 (C-8a), 134.2 (C-30, C-50), 140.4 (C-
6), 143.0 (C-100), 143.7 (C-4a), 161.6 (C-10); HRMS (ESI) m/z calcd for
C23H21N2O3 [M þ H]þ 373. 1552, found 373.1547; IR (KBr, v, cmꢄ1):
3378, 2221, 1508, 1250, 1176, 828; HPLC: 100%, tr 11.7 min; Anal.
(C23H20N2O3) C, H, N.
Synthesized from 5c (1.17 g, 5.54 mmol) according to the general
procedure for synthesis of compounds 6aed; pale yellow crystals,
yield 865 mg (50%); mp 159e162 ꢃC; [
a
]
¼ ꢄ29.7 (c ¼ 0.5, CDCl3);
D
1H NMR (400 MHz, DMSO-d6):
d
(ppm) 4.33 (dd, J ¼ 11.7, 7.3 Hz, 1H,
3-CH2), 4.38 (dd, J ¼ 11.2, 5.7 Hz,1H, CH2O), 4.45 (dd, J ¼ 11.2, 3.7 Hz,
1H, CH2O), 4.62 (dd, J ¼ 11.7, 2.5 Hz,1H, 3-CH2), 4.71e4.79 (m,1H, 2-
CH), 7.14e7.21 (m, 3H, AreH5, AreH2’, AreH6’), 7.75e7.85 (m, 4H,
AreH6, AreH8, AreH3’, AreH5’); 13C NMR (101 MHz, DMSO-d6):
6.13. (S)-4-((6-(benzylamino)-2,3-dihydro-1,4-benzodioxin-2-yl)
methoxy)benzonitrile (8b)
d
(ppm) 65.0 (C-3), 66.5 (CH2O), 71.4 (C-2), 103.4 (C-40), 112.7 (C-6),
115.7 (C-20, C-60), 117.5, 117.6 (C-5, C-8), 119.0 (CN), 134.2 (C-30, C-50),
141.2 (C-7), 142.5, 149.0 (C-4a, C-8a), 161.4 (C-10); HRMS (ESI) m/z
calcd for C16H13N2O5 [M þ H]þ 313.0824, found 313.0827; IR (KBr, v,
cmꢄ1): 2227, 1605, 1515, 1457, 1350, 1256, 1175, 839; HPLC: 100%, tr
16.0 min; Anal. (C16H12N2O5) C, H, N.
Synthesized from 6b (800 mg, 2.56 mmol) according to the
general procedure for synthesis of compounds 8aed; yellow
crystals, yield 439 mg (46%); mp 88e91 ꢃC; [
a
]
¼ ꢄ32.8 (c ¼ 0.5
D
in CHCl3); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 4.00
(dd, J ¼ 11.4, 7.2 Hz, 1H, 3-CH2), 4.18 (d, J ¼ 5.9 Hz, 2H, NCH2),
4.23e4.35 (m, 3H, 3-CH2, CH2O), 4.44e4.55 (m, 1H, 2-CH), 5.93 (bt,
J ¼ 6.1 Hz, 1H, NH), 6.07 (d, J ¼ 2.6 Hz, 1H, AreH5), 6.15 (dd, J ¼ 8.7,
2.7 Hz, 1H, AreH7), 6.62 (d, J ¼ 8.7 Hz, 1H, AreH8), 7.16 (d,
J ¼ 9.0 Hz, 2H, AreH2’, AreH6’), 7.19e7.26 (m, 1H, Ph), 7.28e7.37
(m, 4H, Ph), 7.78 (d, J ¼ 9.0 Hz, 2H, AreH3’, AreH5’); 13C NMR
6.11. (S)-4-((7-nitro-2,3-dihydro-1,4-benzodioxin-2-yl)methoxy)
benzonitrile (6d)
Synthesized from 5d (1.17 g, 5.54 mmol) according to the
general procedure for synthesis of compounds 6aed; pale yellow
crystals, yield 830 mg (48%); mp 168e171 ꢃC; [
a]
D
¼ þ30.2 (c ¼ 0.5,
(101 MHz, DMSO-d6): d (ppm) 47.0 (PheCH2), 64.2 (C-3), 66.9
CHCl3); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 4.33 (dd, J ¼ 11.7,
(CH2O), 71.4 (C-2), 100.3 (C-5), 103.2 (C-40), 106.3 (C-7), 115.6 (C-20,
C-60), 117.1 (C-8), 119.0 (CN), 126.4 (C-400), 127.1 (C-200, C-600), 128.2
(C-300, C-500), 133.6 (C-8a), 134.2 (C-30, C-50), 140.4 (C-6), 142.7 (C-
100), 143.9 (C-4a), 161.6 (C-10); HRMS (ESI) m/z calcd for C23H21N2O3
[M þ H]þ 373.1552, found 373.1534; IR (KBr, v, cmꢄ1): 2219, 1606,
1504, 1260, 1174, 1045, 830; HPLC: 98.1%, tr 11.7 min; Anal.
(C23H20N2O3) C, H, N.
7.3 Hz, 1H, 3-CH2), 4.38 (dd, J ¼ 11.1, 5.7 Hz, 1H, CH2O), 4.45 (dd,
J ¼ 11.1, 3.7 Hz, 1H, CH2O), 4.62 (dd, J ¼ 11.7, 2.5 Hz, 1H, 3-CH2),
4.68e4.81 (m, 1H, 2-CH), 7.11e7.23 (m, 3H, AreH5, AreH2’, AreH6’),
7.74e7.85 (m, 4H, AreH6, AreH8, AreH3’, AreH5’); 13C NMR
(101 MHz, DMSO-d6):
d (ppm) 64.9 (C-3), 66.5 (CH2O), 71.4 (C-2),
103.4 (C-40), 112.7 (C-6), 115.7 (C-20, C-60), 117.5, 117.6 (C-5, C-
8), 119.0 (CN), 134.2 (C-30, C-50), 141.2 (C-7), 142.5, 149.0 (C-4a, C-
8a), 161.4 (C-10); HRMS (ESI) m/z calcd for C16H13N2O5 [M þ H]þ
313.0824, found 313.0833; IR (KBr, v, cmꢄ1): 2225, 1600,
1504, 1349, 1251, 820; HPLC: 100%, tr 16.0 min; Anal. (C16H12N2O5)
C, H, N.
6.14. (R)-4-((7-(benzylamino)-2,3-dihydro-1,4-benzodioxin-2-yl)
methoxy)benzonitrile (8c)
Synthesized from 6c (800 mg, 2.56 mmol) according to the
general procedure for synthesis of compounds 8aed; yellow
6.12. (R)-4-((6-(benzylamino)-2,3-dihydro-1,4-benzodioxin-2-yl)
methoxy)benzonitrile (8a). General procedure for synthesis of
compounds 8aed
crystals, yield 420 mg (44%); mp 111e114 ꢃC; [
a
]
¼ ꢄ42.5
D
(c ¼ 0.8, CDCl3); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 4.00
(dd, J ¼ 11.5, 7.2 Hz, 1H, 3-CH2), 4.18 (d, J ¼ 4.8 Hz, 2H, NCH2),
4.22e4.35 (m, 3H, 3-CH2, CH2O), 4.43e4.57 (m, 1H, 2-CH), 5.93
(bt, J ¼ 5.6 Hz, 1H, NH), 6.07 (d, J ¼ 2.6 Hz, 1H, AreH8), 6.15 (dd,
J ¼ 8.7, 2.6 Hz, 1H, AreH6), 6.62 (d, J ¼ 8.7 Hz, 1H, AreH5), 7.16 (d,
J ¼ 9.0 Hz, 2H, AreH2’, AreH6’), 7.19e7.25 (m, 1H, Ph), 7.27e7.38
(m, 4H, Ph), 7.78 (d, J ¼ 9.0 Hz, 2H, AreH3’, AreH5’); 13C NMR
To a solution of compound 6a (800 mg, 2.56 mmol) in 100 mL
MeOH, 10% Pd/C (80 mg) was added and the mixture was stirred in
a hydrogenator at 25 bar for 1 h till the reaction was completed. The
catalyst was filtered off and the solvent was evaporated in vacuo to
yield 720 mg (100%) of 7a which was used in the next step without
purification. To a solution of the crude amine 7a (722 mg,
2.56 mmol) in methanol (50 mL) containing molecular sieves under
argon atmosphere, benzaldehyde (406 mg, 3.83 mmol) was added
and the mixture was stirred at room temperature for 12 h, until the
aldimine formation was completed. The reaction mixture contain-
ing aldimine in methanol was carefully treated with NaBH4
(154 mg, 4.08 mmol) and stirred for additional 1 h, filtered, and the
solvent was evaporated in vacuo. The crude residue was dissolved
in dichloromethane (50 mL) and washed successively with satu-
rated NaHCO3 solution (3 ꢂ 50 mL) and brine (1 ꢂ 50 mL). The
organic phase was dried over Na2SO4 and the solvent evaporated
under reduced pressure. The oily product was purified by column
chromatography using dichloromethane as eluant to obtain 439 mg
(101 MHz, DMSO-d6):
d (ppm) 47.0 (PheCH2) 64.2 (C-3), 66.7
(CH2O), 71.4 (C-2), 100.3 (C-8), 103.2 (C-40), 106.3 (C-6), 115.6 (C-20,
C-60), 117.1 (C-5), 119.0 (CN), 126.5 (C-400), 127.1 (C-200, C-600), 128.2
(C-300, C-500), 133.6 (C-8a), 134.2 (C-30, C-50), 140.4 (C-7), 142.7 (C-
100), 143.9 (C-4a), 161.6 (C-10); HRMS (ESI) m/z calcd for
C23H21N2O3 [M þ H]þ 373. 1552, found 373.1545; IR (KBr, v, cmꢄ1):
3387, 3050, 2220, 1508, 1262, 1174, 832; HPLC: 98.8%, tr 11.7 min;
Anal. (C23H20N2O3) C, H, N.
6.15. (S)-4-((7-(benzylamino)-2,3-dihydro-1,4-benzodioxin-2-yl)
methoxy)benzonitrile (8d)
Synthesized from 7d (800 mg, 2.56 mmol) according to the
general procedure for synthesis of compounds 8aed; yellow
(46%) of 8a as yellow crystals; mp 144e147 ꢃC; [
a]
D
¼ þ32.4
crystals, yield 430 mg (45%); mp 86e89 ꢃC; [
a
]
¼ þ44.4 (c ¼ 0.8,
D
(c ¼ 0.5, CHCl3); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 4.05 (dd,
CDCl3); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 4.00 (dd, J ¼ 11.4,
J ¼ 11.4, 7.9 Hz, 1H, 3-CH2), 4.18 (s, 2H, NCH2), 4.22e4.36 (m, 3H, 3-
CH2, CH2O), 4.38e4.47 (m, 1H, 2-CH), 5.92 (bs, 1H, NH), 6.09 (d,
J ¼ 2.6 Hz, 1H, AreH5), 6.15 (dd, J ¼ 8.7, 2.6 Hz, 1H, AreH7), 6.62 (d,
J ¼ 8.7 Hz, 1H, AreH8), 7.16 (d, J ¼ 9.0 Hz, 2H, AreH2’, AreH6’), 7.19e
7.2 Hz, 1H, 3-CH2), 4.18 (d, J ¼ 5.7 Hz, 2H, NCH2), 4.24e4.34 (m, 3H,
3-CH2, CH2O), 4.46e4.53 (m, 1H, 2-CH), 5.93 (bt, J ¼ 5.9 Hz, 1H,
NH), 6.07 (d, J ¼ 2.6 Hz, 1H, AreH8), 6.15 (dd, J ¼ 8.7, 2.6 Hz,
1H, AreH6), 6.62 (d, J ¼ 8.7 Hz, 1H, AreH5), 7.16 (d, J ¼ 8.9 Hz, 2H,

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M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
336
AreH2’, AreH6’), 7.18e7.26 (m, 1H, Ph), 7.28e7.37 (m, 4H, Ph), 7.78
(d, J ¼ 8.9 Hz, 2H, AreH3’, AreH5’); 13C NMR (101 MHz, DMSO-d6):
1669, 1606, 1507, 1256, 1172, 1028, 835; HPLC: 100%, tr 17.6 min;
Anal. (C27H24N2O6) C, H, N.
d
(ppm) 47.0 (PheCH2), 64.2 (C-3), 66.9 (CH2O), 71.4 (C-2), 100.3
(C-8), 103.2 (C-40), 106.3 (C-6), 115.6 (C-20, C-60), 117.1 (C-5), 119.0
(CN), 126.4 (C-400), 127.1 (C-200, C-600), 128.2 (C-300, C-500), 133.6 (C-
8a), 134.2 (C-30, C-50), 140.4 (C-7), 142.7 (C-100), 143.9 (C-4a), 161.6
6.18. (R)-ethyl 2-(benzyl(3-((4-cyanophenoxy)methyl)-2,3-dihydro-
1,4-benzodioxin-7-yl)amino)-2-oxoacetate (9c)
(C-10); HRMS (ESI) m/z calcd for C23H21N2O3 [M
þ
H]þ
Synthesized from 8c (420 mg, 1.13 mmol) according to the
general procedure for the synthesis of compounds 9aed; pale
373.1552, found 373.1548; IR (KBr, v, cmꢄ1): 3386, 3059, 2220,
1606, 1509, 1262, 1174, 832; HPLC: 93.6%, tr 11.8 min; Anal.
(C23H20N2O3) C, H, N.
yellow solid, yield 486 mg (91%); mp 43e46 ꢃC; [
a]
¼ ꢄ14.2
D
(c ¼ 0.5, CHCl3); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 0.90 (t,
J ¼ 7.1 Hz, 3H, CH2CH3), 4.00 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.14 (dd,
J ¼ 11.6, 7.3 Hz, 1H, 3-CH2), 4.29 (dd, J ¼ 11.0, 5.8 Hz, 1H, CH2O), 4.37
(dd, J ¼ 11.0, 3.6 Hz, 1H, CH2O), 4.44 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2),
4.55e4.64 (m, 1H, 2-CH), 4.90 (s, 2H, NCH2), 6.62 (dd, J ¼ 8.6, 2.6 Hz,
1H, AreH6), 6.80 (d, J ¼ 2.6 Hz, 1H, AreH8), 6.88 (d, J ¼ 8.6 Hz, 1H,
AreH5), 7.16 (d, J ¼ 9.0 Hz, 2H, AreH2’, AreH6’), 7.17e7.21 (m, 2H,
Ph), 7.25e7.35 (m, 3H, Ph), 7.80 (d, J ¼ 9.0 Hz, 2H, AreH3’, AreH5’);
6.16. (R)-ethyl 2-(benzyl(2-((4-cyanophenoxy)methyl)-2,3-dihydro-
1,4-benzodioxin-6-yl)amino)-2-oxoacetate (9a). General procedure
for the synthesis of compounds 9aed
Ethyl oxalyl chloride (185 mg, 1.35 mmol) was added to a solu-
tion of 8a (420 mg, 1.13 mmol) and triethylamine (136 mg,
1.35 mmol) in dichloromethane (50 mL) and the mixture was
stirred for 2 h. The solvent was removed under reduced pressure,
the residue dissolved in ethyl acetate (50 mL) and washed suc-
cessively with a 10% citric acid solution (3 ꢂ 50 mL), saturated
NaHCO3 solution (3 ꢂ 50 mL) and brine (1 ꢂ 50 mL). The organic
phase was dried over Na2SO4 and the solvent evaporated under
reduced pressure. The oily product was purified by column chro-
matography on silica gel using dichloromethane as eluant to obtain
13C NMR (101 MHz, DMSO-d6):
d (ppm) 13.3 (CH2eCH3), 50.8 (Phe
CH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.6 (CH2O), 71.3 (C-2), 103.3 (C-
40), 115.6 (C-20, C-60), 116.2 (C-6), 117.2 (C-5), 119.0 (CN), 120.7 (C-8),
127.5 (C-400), 127.9 (C-200, C-600), 128.6 (C-300, C-500), 132.5 (C-7), 134.2
(C-30, C-50), 136.1 (C-100), 142.5, 142.8 (C-4a, C-8a), 161.4, 161.5, 162.4
(COeCOO, COeCOO, C-10); HRMS (EI) m/z calcd for C27H25N2O6
[M þ H]þ 473.1713, found 473.1718; IR (KBr, v, cmꢄ1): 2224, 1741,
1669, 1605, 1508, 1257, 1173, 1028, 834; HPLC: 100%, tr 17.5 min;
Anal. (C27H24N2O6) C, H, N.
497 mg (93%) of 9a as pale yellow solid; mp 42e45 ꢃC; [
a]
D
¼ þ28.4
(c ¼ 0.5, CHCl3); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 0.93 (t,
J ¼ 7.1 Hz, 3H, CH2CH3), 4.02 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.13 (dd,
J ¼ 11.6, 7.4 Hz, 1H, 3-CH2), 4.30 (dd, J ¼ 11.0, 5.7 Hz, 1H, CH2O), 4.37
(dd, J ¼ 11.0, 3.7 Hz, 1H, CH2O), 4.44 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2),
4.56e4.64 (m, 1H, 2-CH), 4.90 (s, 2H, NCH2), 6.62 (dd, J ¼ 8.6, 2.7 Hz,
1H, AreH7), 6.78 (d, J ¼ 2.7 Hz, 1H, AreH5), 6.89 (d, J ¼ 8.6 Hz, 1H,
AreH8), 7.15 (d, J ¼ 9.0 Hz, 2H, AreH2’, AreH6’), 7.18e7.22 (m, 2H,
Ph), 7.27e7.37 (m, 3H, Ph), 7.79 (d, J ¼ 9.0 Hz, 2H, AreH3’, AreH5’);
6.19. (S)-ethyl 2-(benzyl(3-((4-cyanophenoxy)methyl)-2,3-dihydro-
1,4-benzodioxin-7-yl)amino)-2-oxoacetate (9d)
Synthesized from 8d (420 mg, 1.13 mmol) according to the
general procedure for the synthesis of compounds 9aed; pale
yellow solid, yield 470 mg (88%); mp 45e48 ꢃC; [
a]
¼ þ12.8
D
(c ¼ 0.5, CHCl3); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 0.93 (t,
13C NMR (101 MHz, DMSO-d6):
d
(ppm) 13.4 (CH2eCH3), 50.8 (Phe
J ¼ 7.1 Hz, 3H, CH2CH3), 4.02 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.13 (dd,
J ¼ 11.6, 7.4 Hz, 1H, 3-CH2), 4.30 (dd, J ¼ 11.0, 5.8 Hz, 1H, CH2O), 4.37
(dd, J ¼ 11.0, 3.7 Hz, 1H, CH2O), 4.44 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2),
4.56e4.64 (m,1H, 2-CH), 4.90 (s, 2H, NCH2), 6.62 (dd, J ¼ 8.6, 2.5 Hz,
1H, AreH6), 6.78 (d, J ¼ 2.5 Hz, 1H, AreH8), 6.89 (d, J ¼ 8.6 Hz, 1H,
AreH5), 7.15 (d, J ¼ 9.0 Hz, 2H, AreH2’, AreH6’), 7.17e7.22 (m, 2H,
Ph), 7.25e7.38 (m, 3H, Ph), 7.79 (d, J ¼ 9.0 Hz, 2H, AreH3’, AreH5’);
CH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.6 (CH2O), 71.3 (C-2), 103.3 (C-
40), 115.6 (C-20, C-60), 116.2 (C-7), 117.4 (C-5), 119.0 (CN), 120.9 (C-8),
127.5 (C-400), 128.0 (C-200, C-600), 128.6 (C-300, C-500), 132.3 (C-6), 134.2
(C-30, C-50), 136.1 (C-100), 142.6, 142.7 (C-4a, C-8a), 161.4, 161.5, 162.4
(COeCOO, COeCOO, C-10); HRMS (ESI) m/z calcd for C27H25N2O6
[M þ H]þ 473.1713, found 473.1706; IR (KBr, v, cmꢄ1): 2224, 1605,
1507, 1252, 1175, 1016, 834; HPLC: 100%, tr 17.6 min; Anal.
(C27H24N2O6) C, H, N.
13C NMR (101 MHz, DMSO-d6):
d (ppm) 13.4 (CH2eCH3), 50.8 (Phe
CH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.6 (CH2O), 71.3 (C-2), 103.3 (C-
40), 115.6 (C-20, C-60), 116.1 (C-6), 117.4 (C-5), 119.0 (CN), 120.9 (C-8),
127.5 (C-400), 128.0 (C-200, C-600), 128.6 (C-300, C-500), 132.3 (C-7), 134.2
(C-30, C-50), 136.1 (C-100), 142.6, 142.7 (C-4a, C-8a), 161.4, 161.5, 162.4
(COeCOO, COeCOO, C-10); HRMS (ESI) m/z calcd for C27H25N2O6
[M þ H]þ 473.1713, found 473.1704; IR (KBr, v, cmꢄ1): 2225, 1741,
1670, 1606, 1508, 1256, 1172, 1029, 835; HPLC: 100%, tr 17.5 min;
Anal. (C27H24N2O6 ꢂ 1/2H2O) C, H, N.
6.17. (S)-ethyl 2-(benzyl(2-((4-cyanophenoxy)methyl)-2,3-dihydro-
1,4-benzodioxin-6-yl)amino)-2-oxoacetate (9b)
Synthesized from 8b (420 mg, 1.13 mmol) according to the
general procedure for the synthesis of compounds 9aed; pale
yellow solid, yield 481 mg (90%); mp 41e44 ꢃC; [
a]
¼ ꢄ27.8
D
(c ¼ 0.5, CHCl3); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 0.90 (t,
J ¼ 7.1 Hz, 3H, CH2CH3), 4.00 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.14 (dd,
J ¼ 11.6, 7.3 Hz, 1H, 3-CH2), 4.29 (dd, J ¼ 11.0, 5.9 Hz, 1H, CH2O), 4.37
(dd, J ¼ 11.0, 3.5 Hz, 1H, CH2O), 4.44 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2),
4.56e4.63 (m,1H, 2-CH), 4.90 (s, 2H, NCH2), 6.62 (dd, J ¼ 8.6, 2.5 Hz,
1H, AreH7), 6.80 (d, J ¼ 2.5 Hz, 1H, AreH5), 6.88 (d, J ¼ 8.6 Hz, 1H,
AreH8), 7.16 (d, J ¼ 9.0 Hz, 2H, AreH2’, AreH6’), 7.17e7.21 (m, 2H,
Ph), 7.23e7.36 (m, 3H, Ph), 7.79 (d, J ¼ 8.9 Hz, 2H, AreH3’, AreH5’);
6.20. (R)-ethyl 2-(benzyl(2-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydro-1,4-benzodioxin-6-yl)amino)-2-oxoacetate trifluoroacetate
(10a). General procedure for synthesis of amidines 10aed
Gaseous HCl was slowly introduced over 30 min into a solution
of the nitrile 9a (480 mg, 1.02 mmol) in anhydrous ethanol (30 mL).
The reaction mixture was closed tightly and stirred for 24 h at room
temperature. The solvent was evaporated in vacuo and the residue
washed 3 times with anhydrous diethyl ether. Obtained iminoether
was dissolved in anhydrous EtOH (30 mL), ammonium acetate (3
eq, 237 mg, 3.06 mmol) was added and the reaction mixture stirred
for 24 h at room temperature. The solvent was evaporated and
crude product was purified by reverse phase column chromatog-
raphy with gradient using methanol/trifluoroacetic acid (40e80%
13C NMR (101 MHz, DMSO-d6):
d (ppm) 13.3 (CH2eCH3), 50.8 (Phe
CH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.6 (CH2O), 71.3 (C-2), 103.3 (C-
40), 115.6 (C-20, C-60), 116.2 (C-7), 117.2 (C-5), 119.0 (CN), 120.7 (C-8),
127.5 (C-400), 127.9 (C-200, C-600), 128.6 (C-300, C-500), 132.5 (C-6), 134.2
(C-30, C-50), 136.1 (C-100), 142.5, 142.8 (C-4a, C-8a), 161.4, 161.5, 162.4
(COeCOO, COeCOO, C-10); HRMS (ESI) m/z calcd for C27H25N2O6
[M þ H]þ 473.1713, found 473.1732; IR (KBr, v, cmꢄ1): 2225, 1741,

ꢁ
M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
337
in 30 min) as eluant. After evaporation of methanol white crystals
were precipitated from trifluoroacetic acid, filtered off and dried to
yield 350 mg (70%) of 10a as a white powder; yield 350 mg (58%);
(101 MHZ, DMSO-d6):
d
(ppm) 13.3 (CH2eCH3), 50.8 (PheCH2), 61.3
(CH2eCH3), 64.4 (C-3), 66.7 (CH2O), 71.3 (C-2), 114.8 (C-20, C-60),
116.2 (C-6), 117.3 (C-5), 120.1 (C-40), 120.8 (C-8), 127.5 (C-400), 127.9
(C-200, C-600), 128.6 (C-300, C-500), 130.2 (C-30, C-50), 132.5 (C-7), 136.1
(C-100), 142.5, 142.8 (C-4a, C-8a), 161.5, 162.3, 162.4, 164.5 (COeCOO,
mp 188e191 ꢃC; [
DMSO-d6):
a
]
D
¼ þ31.4 (c ¼ 0.5, EtOH); 1H NMR (400 MHz,
d
(ppm) 0.93 (t, J ¼ 7.1 Hz, 3H, CH2CH3), 4.02 (q,
1
J ¼ 7.1 Hz, 2H, CH2CH3), 4.14 (dd, J ¼ 11.6, 7.4 Hz, 1H, 3-CH2), 4.32
(dd, J ¼ 11.0, 5.8 Hz, 1H, CH2O), 4.39 (dd, J ¼ 11.0, 3.7 Hz, 1H, CH2O),
4.46 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2), 4.57e4.65 (m, 1H, 2-CH), 4.90
(s, 2H, NCH2), 6.63 (dd, J ¼ 8.6, 2.6 Hz, 1H, AreH7), 6.79 (d,
J ¼ 2.6 Hz, 1H, AreH6), 6.89 (d, J ¼ 8.6 Hz, 1H, AreH8), 7.17e7.24 (m,
4H, Ph, AreH2’, AreH6’), 7.25e7.38 (m, 3H, Ph), 7.83 (d, J ¼ 9.0 Hz,
2H, AreH3’, AreH5’), 8.92 (bs, 2H, NH2), 9.16 (bs, 2H, NHþ2 ); 13C NMR
COeCOO, C-10, C(]NH)NH2), 117.1 (CF3eCOOH, JC,F ¼ 299.2 Hz),
2
158.8 (CF3eCOOH, JC,F ¼ 31.5 Hz); HRMS (ESI) m/z calcd for
C27H28N3O6 [M þ H]þ 490.1978, found 490.1996; IR (KBr, v, cmꢄ1):
3345, 3109, 1742, 1670, 1500, 1197, 843; HPLC: 98.4%, tr 12.7 min;
Anal. (C27H27N3O6 ꢂ CF3COOH) C, H, N.
6.23. (S)-ethyl 2-(benzyl(3-((4-carbamimidoylphenoxy)methyl)-
2,3-dihydro-1,4-benzodioxin-7-yl)amino)-2-oxoacetate (10d)
(101 MHz, DMSO-d6): d (ppm) 13.4 (CH2eCH3), 50.8 (PheCH2), 61.3
(CH2eCH3), 64.4 (C-3), 66.7 (CH2O), 71.3 (C-2), 114.8 (C-20, C-60),
116.2 (C-7), 117.4 (C-5), 120.1 (C-40), 120.9 (C-8), 127.6 (C-400), 128.0
(C-200, C-600), 128.6 (C-300, C-500), 130.2 (C-30, C-50), 132.3 (C-6), 136.1
(C-100), 142.6, 142.7 (C-4a, C-8a), 161.5, 162.3, 162.4, 164.5 (COeCOO,
Synthesized from 9d (480 mg, 1.02 mmol) according to the
general procedure for synthesis of amidines 10aed; white pow-
der, yield 345 mg (56%); mp 188e191 ꢃC; [
a
]
¼ ꢄ12.8 (c ¼ 2.0,
D
1
COeCOO, C-10, C(]NH)NH2), 117,1 (CF3eCOOH, JC,F ¼ 299,3 Hz),
DMSO); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 0.91 (t, J ¼ 7.1 Hz,
2
158,7 (CF3eCOOH, JC,F ¼ 31,5 Hz); HRMS (ESI) m/z calcd for
3H, CH2CH3), 4.00 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.15 (dd, J ¼ 11.6,
7.3 Hz, 1H, 3-CH2), 4.31 (dd, J ¼ 11.0, 5.9 Hz, 1H, CH2O), 4.39 (dd,
J ¼ 11.0, 3.5 Hz, 1H, CH2O), 4.46 (dd, J ¼ 11.6, 2.2 Hz, 1H, 3-CH2),
4.53e4.68 (m, 1H, 2-CH), 4.90 (s, 2H, NCH2), 6.63 (dd, J ¼ 8.6,
2.5 Hz, 1H, AreH6), 6.80 (d, J ¼ 2.5 Hz, 1H, AreH8), 6.89 (d,
J ¼ 8.6 Hz,1H, AreH5), 7.17e7.24 (m, 4H, Ph, AreH2’, AreH6’), 7.26e
7.37 (m, 3H, Ph), 7.83 (d, J ¼ 8.9 Hz, 2H, AreH3’, AreH5’), 8.93 (bs,
2H, NH2), 9.17 (bs, 2H, NHþ2 ); 13C NMR (101 MHz, DMSO-d6):
C27H28N3O6 [M þ H]þ 490.1978, found 490.1967; IR (KBr, v, cmꢄ1):
3299, 3116, 1737, 1667, 1506, 1203, 844; HPLC: 99.0%, tr 12.5 min;
Anal. (C27H27N3O6 ꢂ CF3COOH) C, H, N.
6.21. (S)-ethyl 2-(benzyl(2-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydro-1,4-benzodioxin-6-yl)amino)-2-oxoacetate trifluoroacetate
(10b)
d
(ppm) 13.3 (CH2eCH3), 50.8 (PheCH2), 61.3 (CH2eCH3), 64.4 (C-
Synthesized from 9b (480 mg, 1.02 mmol) according to the
general procedure for synthesis of amidines 10aed; white powder,
3), 66.7 (CH2O), 71.3 (C-2), 114.8 (C-20, C-60), 116.2 (C-6), 117.3 (C-
5), 120.1 (C-40), 120.8 (C-8), 127.5 (C-400), 127.9 (C-200, C-600), 128.6
(C-300, C-500), 130.2 (C-30, C-50), 132.5 (C-7), 136.1 (C-100), 142.5,
142.8 (C-4a, C-8a), 161.5, 162.3, 162.4, 164.5 (COeCOO, COeCOO, C-
10, C(]NH)NH2); HRMS (ESI) m/z calcd for C27H28N3O6 [M þ H]þ
490.1978, found 490.1987; IR (KBr, v, cmꢄ1): 3342, 3110, 1742,
1670, 1499, 1196, 844; HPLC: 98.4%, tr 12.7 min; Anal.
(C27H27N3O6 ꢂ CF3COOH) C, H, N.
yield 325 mg (53%); mp 190e193 ꢃC; [
a
]
¼ ꢄ32.0 (c ¼ 0.5 in EtOH);
D
1H NMR (400 MHz, DMSO-d6):
d
(ppm) 0.93 (t, J ¼ 7.1 Hz, 3H,
CH2CH3), 4.02 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 4.14 (dd, J ¼ 11.6, 7.4 Hz,
1H, 3-CH2), 4.32 (dd, J ¼ 11.0, 5.7 Hz, 1H, CH2O), 4.39 (dd, J ¼ 11.0,
3.6 Hz, 1H, CH2O), 4.46 (dd, J ¼ 11.6, 2.4 Hz, 1H, 3-CH2), 4.57e4.66
(m, 1H, 2-CH), 4.91 (s, 2H, NCH2), 6.63 (dd, J ¼ 8.6, 2.7 Hz, 1H,
AreH7), 6.79 (d, J ¼ 2.7 Hz, 1H, AreH5), 6.89 (d, J ¼ 8.6 Hz, 1H, Are
H8), 7.16e7.25 (m, 4H, Ph, AreH2’, AreH6’), 7.27e7.37 (m, 3H, Ph),
7.83 (d, J ¼ 9.0 Hz, 2H, AreH3’, AreH5’), 8.92 (bs, 2H, NH2), 9.16 (bs,
6.24. (R)-2-(benzyl(2-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydro-1,4-benzodioxin-6-yl)amino)-2-oxoacetic acid (11a).
General procedure for synthesis of compounds 11aed
2H, NHþ2 ); 13C NMR (101 MHz, DMSO-d6):
d (ppm) 13.4 (CH2eCH3),
50.8 (PheCH2), 61.3 (CH2eCH3), 64.4 (C-3), 66.7 (CH2O), 71.3 (C-2),
114.8 (C-20, C-60), 116.2 (C-7), 117.4 (C-5), 120.1 (C-40), 120.9 (C-8),
127.6 (C-400), 128.0 (C-200, C-600), 128.6 (C-300, C-500), 130.2 (C-30, C-50),
132.3 (C-6), 136.1 (C-100), 142.6, 142.7 (C-4a, C-8a), 161.5,
162.3, 162.4, 164.5 (COeCOO, COeCOO, C-10, C(]NH)NH2), 117.1
To a solution of 10a (150 mg, 0.31 mmol) in a mixture of
tetrahydrofuran (3 mL) and methanol (1 mL), 1 M LiOH (1.50 mL,
1.50 mmol) was added and the reaction mixture stirred at room
temperature for 1 h. The solvents was evaporated in vacuum and
the resulting aqueous solution neutralized with 0.1% trifluoro-
acetic acid until the product started to precipitate (pH ¼ 2). The
product was filtered off and dried to obtain 79 mg (68%) of 11a as
1
2
(CF3eCOOH, JC,F ¼ 299.2 Hz), 158.8 (CF3eCOOH, JC,F ¼ 31.5 Hz);
HRMS (ESI) m/z calcd for C27H28N3O6 [M þ H]þ 490.1978, found
490.1963; IR (KBr, v, cmꢄ1): 3298, 3122, 1737, 1665, 1506, 1203, 843;
HPLC: 100%, tr 12.4 min; Anal. (C27H27N3O6 ꢂ CF3COOH ꢂ 1/9H2O)
C, H, N.
a white powder; mp 290e293 ꢃC; [
a
]
¼ þ59.6 (c ¼ 2.0, DMSO);
D
1H NMR (400 MHz, DMSO-d6):
d
(ppm) 4.14 (dd, J ¼ 11.6, 7.6 Hz,
6.22. (R)-ethyl 2-(benzyl(3-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydro-1,4-benzodioxin-7-yl)amino)-2-oxoacetate triluoroacetate
(10c)
1H, 3-CH2), 4.33 (dd, J ¼ 11.0, 5.6 Hz, 1H, CH2O), 4.40 (dd, J ¼ 11.0,
3.7 Hz, 1H, CH2O), 4.46 (dd, J ¼ 11.6, 2.3 Hz, 1H, 3-CH2), 4.57e4.66
(m, 1H, 2-CH), 4.88 (s, 2H, NCH2), 6.67 (dd, J ¼ 8.6, 2.5 Hz, 1H, Are
H7), 6.80 (d, J ¼ 2.5 Hz, 1H, AreH5), 6.89 (d, J ¼ 8.6 Hz, 1H, Are
H8), 7.18e7.24 (m, 4H, Ph, AreH2’, AreH6’), 7.27e7.35 (m, 3H,
Ph), 7.83 (d, J ¼ 8.9 Hz, 2H, AreH3’, AreH5’), 9.04 (bs, 2H, NH2),
Synthesized from 9c (480 mg, 1.02 mmol) according to the
general procedure for synthesis of amidines 10aed; white powder,
yield 335 mg (54%); mp 185e188 ꢃC; [
a]
¼ þ11.8 (c ¼ 2.0, DMSO);
9.17 (bs, 1H, NH); 13C NMR (101 MHz, DMSO-d6):
d (ppm) 49.6
D
1H NMR (400 MHz, DMSO-d6):
d
(ppm) 0.91 (t, J ¼ 7.0 Hz, 3H,
(PheCH2), 64.4 (C-3), 66.7 (CH2O), 71.0 (C-2), 114.7 (C-20, C-60),
115.9 (C-7), 116.6 (C-5), 120.2 (C-40), 120.6 (C-8), 127.0 (C-400),
127.6 (C-200, C-600), 128.3 (C-300, C-500), 129.8 (C-30, C-50), 132.3 (C-
6), 137.7 (C-100), 141.4, 142.0 (C-4a, C-8a), 162.0, 163.2, 164.0, 164.6
(COeCOO, COeCOO, C-10, C(]NH)NH2); HRMS (ESI) m/z calcd for
C25H24N3O6 [M þ H]þ 462.1665, found 462.1674; IR (KBr, v,
cmꢄ1): 3368, 1609, 1503, 1255, 844; HPLC: 96.1%, tr 9.5 min; Anal.
(C25H23N3O6 ꢂ 1/3CF3COOH) C, H, N.
CH2CH3), 4.00 (q, J ¼ 7.0 Hz, 2H, CH2CH3), 4.15 (dd, J ¼ 11.6, 7.2 Hz,
1H, 3-CH2), 4.31 (dd, J ¼ 11.0, 6.0 Hz, 1H, CH2O), 4.39 (dd, J ¼ 11.0,
3.4 Hz, 1H, CH2O), 4.46 (dd, J ¼ 11.6, 2.2 Hz, 1H, 3-CH2), 4.58e4.64
(m, 1H, 2-CH), 4.90 (s, 2H, NCH2), 6.63 (dd, J ¼ 8.7, 2.5 Hz, 1H, Are
H6), 6.80 (d, J ¼ 2.5 Hz, 1H, AreH8), 6.89 (d, J ¼ 8.7 Hz, 1H, AreH5),
7.15e7.38 (m, 7H, Ph, AreH2’, AreH6’), 7.83 (d, J ¼ 8.9 Hz, 2H, Are
H3’, AreH5’), 8.91 (bs, 2H, NH2), 9.17 (bs, 2H, NHþ2 ); 13C NMR

ꢁ
M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
338
6.25. (S)-2-(benzyl(2-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydro-1,4-benzodioxin-6-yl)amino)-2-oxoacetic acid (11b)
[M þ H]þ 462.1665, found 462.1684; IR (KBr, v, cmꢄ1): 3309,
1609, 1492, 1262, 839; HPLC: 96.2%, tr 9.7 min; Anal.
(C25H23N3O6 ꢂ 1/11CF3COOH) C, H, N.
Synthesized from 10b (150 mg, 0.25 mmol) according to the
general procedure for synthesis of compounds 11aed; white
6.28. Docking studies
powder, yield 69 mg (60%); mp 294e297 ꢃC; [
a]
¼ ꢄ56.8 (c ¼ 2.0
D
in DMSO); 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 3.91 (dd, J ¼ 11.2,
The binding modes of the ligands to both targets were studied
by Autodock 4.0 [53]. The energetically most favorable con-
formation was found for each stereoisomer with HyperChem, using
the semiempirical method MNDO. All amide bonds had trans
configuration and were marked as non-rotatable. Compounds were
protonated on the basic center (amidino, or amino group) and were
assigned with a negative charge on the acidic center (carboxylic
group). The crystal structures of thrombin (PDB entry code: 1KTS)
[48] and GP IIb/IIIa (PDB entry code: 2VDM) [49] were extracted
from the Brookhaven Protein Database. The PDB crystal structures
were prepared for docking by removing water molecules and li-
gands. In the case of 2VDM, the magnesium ion in the active site
was retained and manually assigned a charge of þ2 and solvation
parameters. The ligands were docked into a restricted box centered
in the active site (22 Å ꢂ 22 Å ꢂ 22 Å). For the global search using
the LGA (Lamarckian genetic algorithm), the size of the initial
random population was 250 individuals, the maximal number of
energy evaluations 1.25 ꢂ 106, the maximal number of generations
500, the number of top individuals that survived into the next
generation (the elitism) 1, the probability that a gene would un-
dergo a random change 0.02, and the crossover probability 0.80.
The average of the worst energy was calculated over a window of 10
generations. For a purely local search, the pseudo Solis and Wets
method was used, whereas the Solis and Wets method was used for
the LGA part of the local search. The parameters used for local
search in both cases were a maximum of 1000 iterations per local
search, the probability of performing a local search on an individual
was 1.0, the maximal number of consecutive successes or failures
before doubling or halving the step size of the local search was 4,
and the lower bound on the step size, 0.01, was the termination
criterion for the local search. A total of 250 dockings were per-
formed, and ligands with RMSD less than 1 were joined in clusters.
The ligand with the lowest docked energy was chosen for inter-
preting the docking results.
7.1 Hz, 1H, 3-CH2), 3.97 (dd, J ¼ 10.1, 5.9 Hz, 1H, CH2O), 4.08 (dd,
J ¼ 10.1, 5.5 Hz, 1H, CH2O), 4.31 (dd, J ¼ 11.2, 1.4 Hz, 1H, 3-CH2),
4.36e4.44 (m, 1H, 2-CH), 4.83 (d, J ¼ 15.3 Hz, 1H, NCH2), 4.89 (d,
J ¼ 15.3 Hz, 1H, NCH2), 6.73e6.85 (m, 3H, AreH8, AreH7, AreH5),
7.03 (d, J ¼ 8.8 Hz, 2H, AreH2’, AreH6’), 7.17e7.24 (m, 3H, Ph),
7.27e7.33 (m, 2H, Ph), 7.82 (d, J ¼ 8.8 Hz, 2H, AreH3’, AreH5’), 9.01
(bs, 2H, NH2), 9.96 (bs, 1H, NH); 13C NMR (101 MHz, DMSO-d6):
d0(ppm) 50.6 (PheCH2), 64.4 (C-3), 66.8 (CH2O), 71.3 (2-C), 114.9 (C-
2 , C-60), 116.1 (C-7), 117.3 (C-5), 120.1 (C-40), 120.9 (C-8), 127.4 (C-
400), 127.8 (C-200, C-600), 128.5 (C-300, C-500), 130.2 (C-30, C-50), 132.8 (C-
6), 136.3 (C-100), 142.5, 142.6 (C-4a, C-8a), 162.3, 163.0, 164.1, 164.5
(COCOO, COeCOO, C-10, C(]NH)NH2); HRMS (ESI) m/z calcd for
C25H24N3O6 [M þ H]þ 462.1665, found 462.1669; IR (KBr, v, cmꢄ1):
3311, 1610, 1500, 1267, 840; HPLC: 96.6%, tr 9.4 min; Anal.
(C25H23N3O6 ꢂ 1/15CF3COOH) C, H, N.
6.26. (R)-2-(benzyl(3-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydro-1,4-benzodioxin-7-yl)amino)-2-oxoacetic acid (11c)
Synthesized from 10c (150 mg, 0.25 mmol) according to the
general procedure for synthesis of compounds 11aed; white
powder, yield 76 mg (66%); mp 291e294 ꢃC; [
a]
¼ þ16.8 (c ¼ 2.0 in
D
DMSO); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 3.86e3.99 (m, 2H,
3-CH2, CH2O), 4.08 (dd, J ¼ 10.1, 5.3 Hz, 1H, CH2O), 4.30 (dd, J ¼ 11.3,
1.8 Hz, 1H, 3-CH2), 4.35e4.45 (m, 1H, 2-CH), 4.82 (d, J ¼ 15.3 Hz, 1H,
NCH2), 4.90 (d, J ¼ 15.3 Hz, 1H, NCH2), 6.74e6.79 (m, 2H, AreH8,
AreH6), 6.81 (d, J ¼ 8.6 Hz, 1H, AreH5), 7.01 (d, J ¼ 8.8 Hz, 2H,
AreH2’, AreH6’), 7.16e7.26 (m, 3H, Ph), 7.27e7.33 (m, 2H, Ph), 7.83
(d, J ¼ 8.8 Hz, 2H, AreH3’, AreH5’), 9.06 (bs, 2H, NH2), 9.80 (bs, 1H,
NH); 13C NMR (101 MHz, DMSO-d6):
d (ppm) 49.4 (PheCH2), 64.5
(C-3), 65.9 (CH2O), 71.0 (C-2), 114.7 (C-20, C-60), 115.2 (C-8), 116.9
(C-6), 118.9 (C-40), 120.1 (C-5), 126.9 (C-400), 127.3 (C-200, C-600), 128.3
(C-300, C-500), 129.8 (C-30, C-50), 135.5 (C-7), 137.8 (C-100), 141.1, 141.6
(C-4a, C-8a), 161.9, 163.2, 164.0, 164.2 (COeCOO, COeCOO, C-10, C(]
NH)NH2); HRMS (ESI) m/z calcd for C25H24N3O6 [M þ H]þ 462.1665,
found 462.1668; IR (KBr, v, cmꢄ1): 3385, 1609, 1508, 1259, 845;
HPLC: 94.0%, tr 9.7 min; Anal. (C25H23N3O6 ꢂ 2/5CF3COOH ꢂ 2/
5H2O) C, H, N.
6.29. Biochemical evaluation
6.29.1. Enzyme assay for inhibition of serine proteases
The enzymic amidolytic method for determining inhibition of
thrombin, factor Xa and trypsin was based on the spectrophoto-
metric 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. Measurements (spectropho-
tometer, BioTek Synergy H4) were performed in 96-well microtiter
6.27. (S)-2-(benzyl(3-((4-carbamimidoylphenoxy)methyl)-2,3-
dihydro-1,4-benzodioxin-7-yl)amino)-2-oxoacetic acid (11d)
Synthesized from 10d (150 mg, 0.25 mmol) according to the
general procedure for synthesis of compounds 11aed; yield
72 mg (62%); mp 290e293 ꢃC; [
a
]
¼ ꢄ17.5 (c ¼ 2.0 in DMSO); 1H
D
NMR (400 MHz, DMSO-d6):
d
(ppm) 4.13 (dd, J ¼ 11.6, 7.9 Hz, 1H,
plates with a final volume of 200
concentration of 0.5 NIH E/mL with the substrate S-2238 (Chro-
mogenix) at 20 and 40 M final concentration, and factor Xa at the
final concentration of 1 mBAEE E/mL with the substrate S-2222
(Chromogenix) at 100 and 200 M final concentrations.
Trypsin was assayed at a final concentration of 0.5 nkat/mL
using the substrate S-2222 (Chromogenix) at 50 and 100 M final
mL. Thrombin was tested in a final
3-CH2), 4.34 (dd, J ¼ 10.9, 5.6 Hz, 1H, CH2O), 4.40 (dd, J ¼ 10.9,
3.6 Hz, 1H, CH2O), 4.48 (dd, J ¼ 11.6, 2.2 Hz, 1H, 3-CH2), 4.55e
4.65 (m, 1H, 2-CH), 4.88 (s, 2H, NCH2), 6.68 (dd, J ¼ 8.6, 2.5 Hz,
1H, AreH6), 6.80 (d, J ¼ 2.5 Hz, 1H, AreH8), 6.89 (d, J ¼ 8.6 Hz,
1H, AreH5), 7.18e7.37 (m, 7H, Ph, AreH2’, AreH6’), 7.83 (d,
J ¼ 8.9 Hz, 2H, AreH3’, AreH5’), 8.91 (s, 2H, NH2), 9.17 (s, 1H, NH);
m
m
m
13C NMR (101 MHz, DMSO-d6):
d
(ppm) 50.6 (PheCH2), 64.4 (C-
concentrations. Inhibitors were dissolved in DMSO (concentration
of stock solutions, 10 mmol/L) and diluted with distilled water to
3), 66.8 (CH2O), 71.4 (C-2), 114.9 (C-20, C-60), 116.1 (C-8), 117.3 (C-
6), 120.1 (C-40), 120.7 (C-5), 127.4 (C-400), 127.8 (C-200, C-600), 128.5
(C-300, C-500), 130.2 (C-30, C-50), 133.0 (C-7), 136.3 (C-100), 142.5,
142.6(C-4a, C-8a), 162.3, 163.1, 164.2, 164.5 (COeCOO, COeCOO,
C-10, C(]NH)NH2); HRMS (ESI) m/z calcd for C25H24N3O6
concentrations from 0.5 to 100
in 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
m
M. Reaction rates were measured
m
M HBSA
m

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M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
339
[2] J. Ruef, H.A. Katus, New antithrombotic drugs on the horizon, Expert Opin.
Investig. Drugs 12 (2003) 781e797.
[3] R. Castelli, F. Porro, D. Savo, E. Cassinerio, New antithrombotic agents, Pan-
minerva. Med. 48 (2006) 247e256.
[4] E. Schaden, S.A. Kozek-Langenecker, Direct thrombin inhibitors: pharmacol-
ogy and application in intensive care medicine, Intensive Care Med. 36 (2010)
1127e1137.
[5] J.I. Weitz, New oral anticoagulants in development, Thromb. Haemost. 103
(2010) 62e70.
[6] J.P. Piccini, R.D. Lopes, K.W. Mahaffey, Oral factor Xa inhibitors for the pre-
vention of stroke in atrial fibrillation, Curr. Opin. Cardiol. 25 (2010) 312e320.
[7] R.A. Shirk, G.P. Vlasuk, Inhibitors of factor VIIa/tissue factor, Arterioscler.
Thromb. Vasc. Biol. 27 (2007) 1895e1900.
50
m
M of enzyme solution were pipetted into the microtiter wells.
L of chro-
The plate was incubated for 15 min at 25 ꢃC and 50
m
mogenic substrate then added. Absorbance at 405 nm was
measured every 10 s at 25 ꢃC. Measurements were carried out in
triplicate with three concentrations of the inhibitor and two con-
centrations of the substrate. For every combination of concentra-
tions, Ki was calculated from the change of absorbance in the initial,
linear part of the curve according to the method of Cheng and
Prusoff [54] and the final result was given as their average value.
Dabigatran (thrombin, Ki ¼ 6.3 ꢀ 1.1 nM) was used as control.
[8] E.L. Howard, K.C.D. Becker, C.P. Rusconi, R.C. Becker, Factor IXa inhibitors as
novel anticoagulants, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 722e727.
[9] M.P. Gulseth, J. Michaud, E.A. Nutescu, Rivaroxaban: an oral direct inhibitor of
factor Xa, Am. J. Health Syst. Pharm. 65 (2008) 1520e1529.
[10] M. Miura, N. Seki, T. Koike, T. Ishihara, T. Niimi, F. Hirayama, T. Shigenaga,
Y. Sakai-Moritani, T. Kawasaki, S. Sakamoto, M. Okada, M. Ohta,
S.I. Tsukamoto, Potent and selective TF/FVIIa inhibitors containing a neutral P1
ligand, Bioorg. Med. Chem. 14 (2006) 7688e7705.
6.29.2. Inhibition of in vitro binding of fibrinogen to isolated GPIIb/
IIIa and GPV/IIIa receptors
Binding affinities to GPIIb/IIIa and GPV/IIIa receptor were
measured by a solid-phase competitive displacement assay. Human
fibrinogen (100 mg) was dissolved in aqueous NaCl (0.3 M, 5 mL) at
30 ꢃC and then it was diluted with 0.1 M NaHCO3 in water 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 fibri-
nogen solution. The reaction mixture was incubated for 90 min at
30 ꢃC 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%) added to
give the stock solution. Human integrin (10
solution (Calbiochem), 1 L of GPV/IIIa receptor solution (Calbio-
chem)) 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
trac 600) overnight at 4 ꢃC. Nonspecific receptor-binding sites were
blocked with 1% BSA in buffer 2 (200 L/well). Following incubation
for 1 h at room temperature, the plates were washed twice with
buffer 3 (buffer 2 containing 0.1% Tween-20). The potential antag-
onists were serially diluted with buffer 2 containing 0.1% of BSA and
ꢀ ꢀ
[11] A. Kranjc, D. Kikelj, L. Peterlin-Masic, Recent advances in the discovery of
tissue factor/factor VIIa inhibitors and dual inhibitors of factor VIIa/factor Xa,
Curr. Pharm. Des. 11 (2005) 4207e4227.
[12] S. Sander, C.M. White, Ximelagatran e a new oral anticoagulant, Formulary 39
(2004) 398e404.
[13] A.C. Spyropoulos, Brave new world: the current and future use of novel an-
ticoagulants, Thromb. Res. 123 (2008) S29eS35.
[14] G.J. Hankey, J.W. Eikelboom, Dabigatran etexilate a new oral thrombin in-
hibitor, Circulation 123 (2011) 1436e1450.
[15] E. Perzborn, S. Roehrig, A. Straub, D. Kubitza, W. Mueck, V. Laux, Rivaroxaban:
a new oralfactor Xa inhibitor, Arterioscler. Thromb. Vasc. Biol. 30 (2010) 376e381.
[16] A.G.G. Turpie, New oral anticoagulants in atrial fibrillation, Eur. Heart J. 29
(2008) 155e165.
[17] P.L. Gross, J.I. Weitz, New antithrombotic drugs, Clin. Pharmacol. Ther. 86
(2009) 139e146.
[18] C. Patrono, C. Baigent, J. Hirsh, G. Roth, Antiplatelet drugs, Chest 133 (2008)
199Se233S.
[19] N. Mackman, Triggers, targets and treatments for thrombosis, Nature 451
(2008) 914e918.
mL of GPIIb/IIIa receptor
m
mL/well) high-binding microtiter plates (Greiner, Lumi-
m
[20] J.D. Douketis, Combination warfarineASA therapy:whichpatientsshouldreceive
it, which patients should not, and why? Thromb. Res. 127 (2011) 513e517.
[21] R. Morphy, C. Kay, Z. Rankovic, From magic bullets to designed multiple li-
gands, Drug Discov. Today 9 (2004) 641e651.
[22] (a) R. Morphy, Z. Rankovic, Fragments, network biology and designing mul-
tiple ligands, Drug Discov. Today 12 (2007) 156e160;
test solutions added (50
nogen (50 L/well, 1:42 dilution of stock solution in buffer 2 con-
mL/well) together with biotinylated fibri-
m
(b) R. Morphy, Z. Rankovic, Designing multiple ligands e medicinal chemistry
strategies and challenges, Curr. Pharm. Des. 15 (2009) 587e600;
(c) R. Morphy, Z. Rankovic, Designed multiple ligands. An emerging drug
discovery paradigm, J. Med. Chem. 48 (2005) 6523e6543.
taining 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
ꢁ
ꢀ
[23] (a) M. Ilic, D. Kikelj, J. Ilas, Multitarget antithrombotic drugs, Curr. Top. Med.
Chem. 11 (2011) 2834e2848;
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-
(b) U. Trstenjak, D. Kikelj, Multitarget cardiovascular drugs, Curr. Med. Chem.
18 (2011) 2531e2542.
ꢀ
ꢀ
ꢀ
[24] P. Stefanic, M. Anderluh, J. Ilas, J. Mravljak, M.S. Dolenc, M. Stegnar, D. Kikelj,
Toward a novel class of antithrombotic compounds with dual function. Dis-
covery of 1,4-benzoxazin-3(4H)-one derivatives possessing thrombin inhib-
itory and fibrinogen receptor antagonistic activities, J. Med. Chem. 48 (2005)
3110e3113.
agnostics, Boehringer Mannheim) (100 mL/well) was added and the
luminescence measured with a BioTek Synergy H4 multimode
research reader three times over 10 min. Positive controls received
no inhibitors, while 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 calculated from the
doseeresponse curve.
ꢀ
ꢀ
ꢀ
[25] 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 function, J. Med. Chem. 51 (2008) 5617e5629.
[26] (a) M. Correia-da-Silva, E. Sousa, B. Duarte, F. Marques, F. Carvalho,
L.M. Cunha-Ribeiro, M.M. Pinto, Polysulfated xanthones: multipathway
development of a new generation of dual anticoagulant/antiplatelet agents,
J. Med. Chem. 54 (2011) 5373e5384;
(b) M. Correia-da-Silva, E. Sousa, B. Duarte, F. Marques, L.M. Cunha-Ribeiro,
M.M. Pinto, Dual anticoagulant/antiplatelet persulfated small molecules, Eur.
J. Med. Chem. 46 (2011) 2347e2358;
Acknowledgments
(c) 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.
This work was supported by the Slovenian Research Agency
(Grant P1-0208) and HungarianeSlovenian bilateral project BI-HU/
ꢀ
ꢁ
09-10-006. Milos Ilic thanks Ad Futura Foundation for scholarship.
The authors thank Professor Roger Pain for critical reading of the
manuscript.
ꢀ
ꢀ
[27] J. Ilas, P.S. Anderluh, M.S. Dolenc, D. Kikelj, Recent advances in the synthesis of
2H-1,4-benzoxazin-3-(4H)-ones and 3,4-dihydro-2H-1,4-benzoxazines, Tet-
rahedron 61 (2005) 7325e7348.
ꢀ ꢀ
[28] L. Peterlin-Masic, D. Kikelj, Arginine mimetics, Tetrahedron 57 (2001) 7073e
7105.
References
ꢀ
[29] J. Ilas, F. Hudecz, H. Süli-Vargha, D. Kikelj, Peptides and pseudopeptides
incorporating D-Phe-Pro-Arg and Arg-Gly-Asp lead sequences as potential
antithrombotic agents, J. Pept. Sci. 14 (2008) 946e953.
[1] A.D. Lopez, C.D. Mathers, M. Ezzati, D.T. Jamison, C.J.L. Murray, Global and
regional burden of disease and risk factors, 2001: systematic analysis of
population health data, Lancet 367 (2006) 1747e1757.
ꢁ
ꢀ
[30] M. Ilic, D. Kikelj, J. Ilas, Fluorinated dual antithrombotic compounds based on
1,4-benzoxazine scaffold, Eur. J. Med. Chem. 50 (2012) 255e263.

ꢁ
M. Ilic et al. / European Journal of Medicinal Chemistry 62 (2013) 329e340
340
ꢁ
ꢀ
[31] M. Ilic, J. Ilas, S. Liekens, P. Mátyus, D. Kikelj, Synthesis and antiproliferative
activity of 2-(([1,2,4]triazolo[4,3-b]-pyridazin-6-yloxy)methyl)-2,4-dimethyl-
3,4-dihydro-2H-benzo[b][1,4]oxazine derivatives, Arkivoc (2011) 298e311.
[32] R. Kikumoto, Y. Tamao, T. Tezuka, S. Tonomura, H. Hara, K. Ninomiya,
A. Hijikata, S. Okamoto, Selective inhibition of thrombin by (2R,4R)-4-methyl-
1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfonyl]-l-arginyl]-2-
piperidinecarboxylic acid, Biochemistry 23 (1984) 85e90.
[42] I. György, S. Antus, A. Blázovics, G. Földiák, Substituent effects in the free
radical reactions of silybin: radiation-induced oxidation of the flavonoid at
neutral pH, Int. J. Radiat. Biol. 61 (1992) 603e609.
[43] D.K. Sharma, I.H. Hall, Hypolipidemic, antiinflammatory, and antineoplastic
activity and cytotoxicity of flavonolignans isolated from Hydnocarpus-
wightiana seeds, J. Nat. Prod. 54 (1991) 1298e1302.
[44] M.T. Vázquez, G. Rosell, M.D. Pujol, Synthesis and antiinflammatory activity of
2,3-dihydro-1,4-benzodioxin methyl carboxylic acids, Farmaco 51 (1996)
215e217.
[45] Y. Harrak, G. Rosell, G. Daidone, S. Plescia, D. Schillaci, M.D. Pujol, Synthesis
and biological activity of new anti-inflammatory compounds containing the
1,4-benzodioxine and/or pyrrole system, Bioorg. Med. Chem. 15 (2007) 4876e
4890.
[46] H. Itazaki, A. Kawasaki, M. Matsuura, M. Ueda, Y. Yonetani, M. Nakamura,
Synthesis of 2,3-dihydro-1,4-benzodioxin derivatives. I. 2-substituted 5 (and
6)-sulfamoyl-2,3-dihydro-1,4-benzodioxins, Chem. Pharm. Bull. 36 (1988)
3387e3403.
[47] M.G. Kelly, J. Kincaid, M. Duncton, K. Sahasrabudhe, S. Janagani, R.B. Upasani,
G. Wu, Y.F. Fang, Z.L. Wei, C. Kaub, J. He, Amide derivatives as ion-channel
ligands and pharmaceutical compositions and methods of using the same,
2006. US 2008/0300243 A1.
[33] S. Okamoto, A. Hijikata, R. Kikumoto, S. Tonomura, H. Hara, K. Ninomiya,
A. Maruyama, M. Sugano, Y. Tamao, Potent inhibition of thrombin by the
newly synthesized arginine derivative No. 805. The importance of stereo-
structure of its hydrophobic carboxamide portion, Biochem. Biophys. Res.
Commun. 101 (1981) 440e446.
[34] M.S. Egbertson, C.T. Chang, M.E. Duggan, R.J. Gould, W. Halczenko,
G.D. Hartman, W.L. Laswell, J.J. Lynch Jr., R.J. Lynch, P.D. Manno, A.M. Naylor,
J.D. Prugh, D.R. Ramjit, G.R. Sitko, R.S. Smith, L.M. Turchi, G. Zhang, Non-
peptide fibrinogen receptor antagonists. 2. Optimization of a tyrosine tem-
plate as a mimic for Arg-Gly-Asp, J. Med. Chem. 37 (1994) 2537e2551.
[35] S.R. Nagarajan, B. Devadas, J.W. Malecha, H.F. Lu, P.G. Ruminski, J.G. Rico,
T.E. Rogers, L.D. Marrufo, J.T. Collins, H.P. Kleine, M.K. Lantz, J. Zhu, N.F. Green,
M.A. Russell, B.H. Landis, L.M. Miller, D.M. Meyer, T.D. Duffin, V.W. Engleman,
M.B. Finn, S.K. Freeman, D.W. Griggs, M.L. Williams, M.A. Nickols, J.A. Pegg,
K.E. Shannon, C. Steininger, M.M. Westlin, G.A. Nickols, J.L. Keene, R-isomers of
Arg-Gly-Asp (RGD) mimics as potent aVb3 inhibitors, Bioorg. Med. Chem. 15
(2007) 3783e3800.
[48] N.H. Hauel, H. Nar, H. Priepke, U. Ries, J.M. Stassen, W. Wienen, Structure-
based design of novel potent nonpeptide thrombin inhibitors, J. Med. Chem.
45 (2002) 1757e1766.
[36] We have recently described the preparation of racemic 1,4-benzodioxine
[49] T. Xiao, J. Takagi, B.S. Coller, J.H. Wang, T.A. Springer, Structural basis for
allostery in integrins and binding to fibrinogen-mimetic therapeutics, Nature
432 (2004) 59e67.
[50] M. Comini, C. Pozzoli, M. Incerti, D. Rossi, S. Collina, O. Azzolina, Di E. Vittorio,
G. Morini, E. Poli, Stereospecific effects of benzo[d]isothiazolyloxypropanol-
ꢁ
ꢀ
ꢀ
compounds in M. Ilic, J. Ilas, P. Dunkel, P. Mátyus, A. Bohác, S. Liekens,
D. Kikelj, Novel 1,4-benzoxazine and 1,4-benzodioxine inhibitors of angio-
genesis, Eur. J. Med. Chem. 58 (2012) 160e170.
[37] A. Wykretowicz, P. Guzik, K. Wysocki, Doxazosin in the current treatment of
hypertension, Expert Opin. Pharmacol. 9 (2008) 625e633.
amine derivatives at
b-adrenoceptors: synthesis, chiral resolution, and bio-
[38] A. Pietrangelo, F. Borella, G. Casalgrandi, G. Montosi, D. Ceccarelli, D. Gallesi,
F. Giovannini, A. Gasparetto, A. Masini, Antioxidant activity of silybin in vivo
during long-term iron overload in rats, Gastroenterology 109 (1995) 1941e
1949.
logical activity in vitro, Chirality 21 (2009) 284e291.
[51] J.M. Klunder, S.Y. Ko, K.B. Sharpless, Asymmetric epoxidation of allyl alcohol:
efficient routes to homochiral
(1986) 3710e3712.
b-adrenergic blocking agents, J. Org. Chem. 51
[39] K. Rácz, J. Fehér, G. Csomós, I. Varga, R. Kiss, E. Glaz, An antioxidant drug,
silibinin, modulates steroid secretion in human pathological adrenocortical
cells, J. Endocrinol. 124 (1990) 341e345.
[52] N. Buizer, C.G. Kruse, M. Van der Laan, G. Langrand, G.J.M. Van Scharrenburg,
M.C. Snoek, Enzymatic Process for the Stereoselective Preparation of a Hetero-
bicyclic Alcohol Enantiomer (1994). EP0605033 (A1).
[40] L. Cavallini, A. Bindoli, N. Siliprandi, Comparative evaluation of anti-
peroxidative action of silymarin and other flavonoids, Pharmacol. Res. Com-
mun. 10 (1978) 133.
[53] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, Autodock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 16 (2009) 2785e2791.
[41] I. Altorjay, L. Dalmi, B. Sári, S. Imre, G. Balla, The effect of silibinin (Legalon) on
the free radical scavenger mechanisms of human erythrocytes in vitro, Acta
Physiol. Hung 80 (1992) 375e380.
[54] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (Ki) and
the concentration of inhibitor which causes 50% inhibition (IC50) of an
enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.