European Journal of Medicinal Chemistry 110 (2016) 181e194
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, evaluation and structure-activity relationship of new
3-carboxamide coumarins as FXIIa inhibitors
goire Rondelet b, Eduard Dolusi
Charlotte Bouckaert a, Silvia Serra a, Gre
c c,
a, Raphae
€l Fre
de
rick d, Lionel Pochet a, *
Johan Wouters b, Jean-Michel Dogne
a
Department of Pharmacy, Namur Medicine & Drug Innovation Center (NAMEDIC), Namur Research Institute for Life Sciences (NARILIS), University of
Namur, 61, Rue de Bruxelles, 5000 Namur, Belgium
b
Department of Chemistry, Namur Medicine & Drug Innovation Center (NAMEDIC), Namur Research Institute for Life Sciences (NARILIS), University of
Namur, Rue de Bruxelles, 61, 5000 Namur, Belgium
c
Department of Organic Chemistry, Namur Medicine & Drug Innovation Center (NAMEDIC), Namur Research Institute for Life Sciences (NARILIS), University
of Namur, 61, Rue de Bruxelles, 5000 Namur, Belgium
d
Medicinal Chemistry Research Group (CMFA), Louvain Drug Research Institute (LDRI), Universit
e Catholique de Louvain, 73, Avenue E. Mounier, 1200
Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 August 2015
Received in revised form
15 December 2015
Accepted 15 January 2016
Available online 16 January 2016
Inhibitors of the coagulation factor XIIa (FXIIa) are attractive to detail the roles of this protease in hemostasis and thrombosis, to suppress artifact due to contact pathway activation in blood coagulation
assays, and they are promising as antithrombotic therapy. The 3-carboxamide coumarins have been
previously described as small-molecular-weight FXIIa inhibitors. In this study, we report a structureactivity relationship (SAR) study around this scaffold with the aim to discover new selective FXIIa inhibitors with an improved physico-chemical profile. To better understand these SAR, docking experiments were undertaken. For this purpose, we built an original hybrid model of FXIIa. This model has the
advantage to gather the best features from the recently published crystal structure of FXIIa in its
zymogen form and a more classical homology model. Results with the hybrid model are encouraging as
they help understanding the activity and selectivity of our best compounds.
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Coumarins
Factor XIIa
Factor XII
FXII
Antithrombotic agents
Benzopyrans
1. Introduction
The blood coagulation system is essential for limiting blood loss
at sites of vascular injury (hemostasis), but excessive and uncontrolled activation leads to thrombotic complications such as
myocardial infarction or deep venous thrombosis. Since thrombotic
disorders are a major cause of morbidity and mortality [1], extensive research has been made in the field of anticoagulation therapy
with the hope to tackle these life-threatening diseases. Currently,
Abbreviations: ADME, absorption, distribution, metabolism and excretion; FXIIa,
activated coagulation factor XII; FXa, activated coagulation factor X; THR, thrombin;
FVIIa, activated factor VII; HPLC-UV, high performance liquid chromatography
coupled with ultraviolet detector; (plasma) kall, (plasma) kallikrein; PDB, Protein
DataBase; p-NA, para-nitroaniline; SAR, structure-activity relationship; TF, tissue
factor; t-PA, tissue plasminogen activator; u-PA, urokinase plasminogen activator.
* Corresponding author.
E-mail address: [email protected] (L. Pochet).
http://dx.doi.org/10.1016/j.ejmech.2016.01.023
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
the most commonly used anticoagulants act by inhibiting thrombin
or factor Xa (FXa), or by lowering the plasma levels of the precursors of these key enzymes [2]. While having largely proven its
antithrombotic efficacy, the current anticoagulant therapy is associated with an unavoidable bleeding risk. Indeed, existing drugs
target key components of the coagulation cascade and they do not
distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis [2].
Coagulation factor XII (FXII) is a trypsin-like serine protease able
to trigger the intrinsic pathway of coagulation through the contact
with negatively charged surfaces (reaction known as the contact
activation). In contrast with thrombin and FXa, FXII has long been
neglected as an attractive target since it was believed to have no
function in hemostasis. This was supported by the clinical observation showing that its deficiency does not lead to bleeding tendency in humans [2,3]. However, investigations with FXII-null mice
completely challenged this dogma. Indeed, it was shown that FXIIdeficient mice are protected against thrombosis without suffering
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C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
from spontaneous or prolonged injury-related bleedings [4e9].
This raised the possibility to design safer antithrombotic agents
without hemostatic defects.
So far, different types of inhibitors of FXII or its activated form
(FXIIa) have been developed [10] including proteins [11e13], synthetic peptides [14], antibodies [15,16] or antisense nucleotides
[17]. Of interest, all of them showed promising results in in vivo
models of thrombosis. Indeed, treated animals were protected
against thrombosis without showing a bleeding phenotype [2].
Given the diversity of inhibitors and thrombosis models investigated, it can be assumed that inhibiting FXII or FXIIa is an innovative strategy to develop antithrombotic agents that would offer
dissociation between hemostasis and thrombosis.
Beyond a safer bleeding profile, such agents could afford new
clinical benefits. Indeed, the leading perspective for the FXII or
FXIIa inhibition is the prevention of contact-mediated thrombosis
induced by medical devices such as catheter or cardiopulmonary
bypass system [16e18]. This statement is supported by studies with
Corn Trypsin Inhibitor (CTI) [18], antisense nucleotides [17] or the
3F7 antibody [16]. More recently, it has been stated that strategies
that target FXII or FXIIa would be of valuable interest in patients
with mechanical heart valves since thrombosis in these patients is
driven by the contact pathway [19].
For acute or sub-chronic applications (i.e. catheter thrombosis,
cardiopulmonary bypass, knee or hip replacement surgery), the
parenteral administration of proteins, peptides, antibodies or RNAderived products would be suitable. In contrast, for chronic indications as encountered in patients exposed to mechanical heart
valves or ventricular assisted-devices, the development of smallmolecular weight inhibitors that would be taken orally is preferable. Coumarin derivatives were previously described by our group
as serine proteases inhibitors [20e24] and the 3-carboxamide
coumarins were especially designed as small-molecular-weight
FXIIa inhibitors [25]. Unfortunately, these compounds lacked of
activity in an in vivo model of thrombosis [26] probably due to poor
pharmacokinetics and/or weak potency. Based on this, we aimed in
the present study to modulate the coumarin scaffold in an effort to
obtain new selective FXIIa inhibitors with optimized physicochemical properties. Moreover, we analyzed the interactions between the compounds and the target in order to better understand
the structure-activity-relationships. Since the three-dimensional
structure of FXIIa is available in a zymogen configuration, we
built an original “hybrid model” of FXIIa to sustain our docking
experiments.
2. Results and discussion
2.1. Chemistry
In our previous study, we identified compound 1 (Fig. 1) as a
promising starting point due to its activity and selectivity [25]. This
compound is a 3-phenylamide coumarin substituted by a 6-chloro8-bromo group. The amide linker between the coumarin and the
side chain in the 3-position and the absence of a chloromethyl
function in the 6-position undoubtedly contribute to the selectivity
of this compound towards related serine proteases such as
thrombin (THR), factor VIIa (FVIIa)/tissue factor (TF) complex, factor
Xa (FXa) and plasma kallikrein (Kall) that require these substitution
patterns for inhibition [25]. Based on these considerations, we
performed modulations on the 3-carboxamide coumarin scaffold in
the 3,6-positions (Fig. 1). These latter aimed to afford selective FXIIa
inhibitors with improved physico-chemical properties. To achieve
this, two series (series A and B) were designed. In the A-series, a
basic group (eNR2) was inserted on either positions while in the Bseries, an oxygen-containing group (eOH or eCOOH) was
introduced.
Compounds of the A-series substituted with various amines in
the 6-position were obtained from the starting material 2 (Scheme
1). This compound was synthesized according to a previously
described procedure [25]. In this series, the phenyl function found
in compound 1 was kept in the 3-position. Compound 3 was obtained by a Delepine's reaction [27] whereas compounds 4e7 were
synthesized following nucleophilic substitution reactions with the
appropriate amines.
Modulations of the 3-position with a basic group (16e22) are
depicted on Scheme 2. Compounds 8e10 were obtained as previously reported by a Knoevenagel-type reaction using the appropriate and commercially available salicylaldehyde [28,29].
Saponification of these compounds afforded the 3-carboxylic acid
derivatives (11e13). Treatment of compound 13 with thionyl
chloride provided the acyl chloride (14) that was further reacted
with the mono N-Boc-protected ethylenediamine to give 15.
Deprotection of the latter in TFA afforded compound 16 [22,28,29].
For compounds 17e22, pivaloyl chloride was preferred as chlorinating agent [30] because it speeded up the amide synthetic route.
Briefly, the 6,8-halo-2-oxo-2H-1-benzopyran-3-carboxylic acids
(11e13) were reacted with triethylamine and pivaloyl chloride in
dichloromethane to form the reactive acyl halide intermediates
that were further reacted with the appropriate amine to afford the
desired target compounds 17e22.
The same synthetic sequence with pivaloyl chloride as chlorinating reagent allowed the synthesis of compounds 23e28 from
compound 13 (Scheme 2) that possess a hydroxyl or carboxylic acid
group in the 2-, 3-, 4-position with or without a chlorine atom in
the 4- or 5-position as substituents on the lateral phenylamide.
Compounds with a hydroxyl function in the 6-position were also
prepared (Scheme 3). The first step consisted in the synthesis of
N,N0 -diphenyl malonamide 30 by reaction of commercial diethyl
malonate 29 with aniline under microwaves irradiations [31]. Then,
a Knoevenagel reaction between compound 30 and the 5(hydroxymethyl)-salicylaldehyde [22] 31 afforded compound 32.
2.2. Physico-chemical properties
Small-molecular-weight inhibitors of FXIIa that could be taken
orally would be ideal for thrombosis prevention in patients with
mechanical heart valves. With that application in mind, we decided
to evaluate in silico the absorption profile of our compounds at the
very early stage of their development. To this end, we preconized an
evaluation of the absorption characteristics of our compounds using the Traffic Lights system elaborated by Lobell et al. [32] with a
view to prioritize our compounds with regard to potential ADMErelated liabilities. The algorithm developed by Lobell (see
Supplementary material for more information) proposes a set of
‘Traffic Lights’ (TLs) [32] based on five physico-chemical properties:
(i) molecular size as assessed by MW corrected for halogens, (ii)
lipophilicity, solubility at pH 6.5, (iv) polarity as assessed by the
polar surface area (PSA), and (v) number of rotatable bonds. Taken
together, the TL's values afford an in silico oral PhysChem score
which ranges from 0 to 10. A low score means that physicochemical properties of the compound are more favorable for the
development of an orally administered drug [32,33].
As observed in Table 1, introduction of a basic group (series A) on
the 3-carboxamide-coumarin scaffold improves the oral PhysChem
score compared to compound 1 whose score is 3. Indeed, nine
compounds from the A-series (3, 6, 16e22) have an oral PhysChem
score of 0 and three have a score of 1 (4, 5, 7). The PhysChem score
of 1 is explained by a moderate Log P and solubility at intestinal pH
(except for compound 17 which has an optimal Log P but a poor
solubility). Modulations in the B-series were focused on the
C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
183
Fig. 1. The starting compound 1 and modulations on positions 3 or 6 of the 3-carboxamide scaffold in series A and in series B.
2.3. Biological profile
Scheme 1. Introduction of a basic group in position 6 of the 3-carboxamide coumarin
scaffold. Reagents and conditions: (i) Hexamethylenetetramine/CHCl3, 70 C, 18 h, then
HCl 6 M/ethanol/H2O, 90 C, 90 min; (ii) ReNH2, ethanol, microwave (100 C, 10 min).
The newly synthesized compounds were first screened on FXIIa
at a single concentration of 50 mM (Tables 1 and 2) and the inhibitory potency (IC50) was determined for compounds displaying at
least 85% inhibition at this concentration. Compounds 23, 24 and 27
revealed to be the three most active derivatives with IC50 of 5, 8 and
7 mM, respectively (Table 3). These data suggest that modulation
with an amino function (3e22) does not afford active compounds
in contrast with the introduction of an oxygen-based group which
yields six active compounds (23e27). Interestingly, the three most
active compounds (23, 24, 27) all have a 2-hydroxyphenyl substituent at the 3-position of the 3-carboxamide coumarin scaffold.
Then, the selectivity of the compounds was evaluated by
investigating the compounds on thrombin, activated factor X (FXa),
complex tissue factor/activated factor VII (TF/FVIIa) and plasma
kallikrein at a concentration of 100 mM. All active compounds on
FXIIa have no inhibition at this concentration against other serine
proteases suggesting they are selective (Table 3).
2.4. Molecular modeling
introduction of an oxygen-containing group on the coumarin
scaffold. When a hydroxymethyl group is inserted in the 6-position
(32), the PhysChem score reaches a value of 0 (Table 2). For compounds 23e28, a hydroxyl- or a carboxyl group was introduced as
substituent on the phenyl side chain. This modulation does not
improve the PhysChem score compared to compound 1 (Table 2).
Moderate Log P and poor solubility (except for compound 28 which
has a high log P and a moderate solubility) are responsible of the
oral PhysChem score of 3.
Solubility is an important factor in the absorption process [34]
and in our series it seems to be the most influencing factor on
the oral PhysChem score. However, taken together, these results are
consistent with a potential oral administration. Indeed, Lobell et al.
observed that their hits (from HTS) had an average oral PhysChem
score of 4.1 while 90% of leads or marketed drugs showed score
lower than 5 [32].
To further explore the interactions between our coumarins and
FXIIa, we carried out molecular modeling experiments. This
implied the elaboration of a 3D model of FXIIa to perform docking
experiments. Regarding the numbering of residues, it should be
mentioned that in the following sections, residues will be
numbered according to the nomenclature of chymotrypsin
(Supplementary material).
2.4.1. The hybrid FXIIa model
Although FXII is a protein known since almost fifty years, the
crystal structure of its catalytic light chain has only been recently
published [35]. In fact, two structures, namely FXIIac and FXIIc
obtained using two different recombinant FXII proteins, were reported by Pathak et al. (PDB: 4XE4 and 4XDE, respectively) [35].
In our study, as we aimed to investigate the interactions and
structure-activity relationships between FXIIa and our derivatives,
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C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
Scheme 2. Introduction of a basic group (16e22) or an oxygen-based group in position 3 (23e28) of the 3-carboxamide coumarin scaffold. Reagents and conditions: (i) NaOH 10%
(w/v), reflux, 30 min, then HCl 6 M; (ii) SOCl2, reflux, 3 h; N-Boc-ethylenediamine/dioxane, room temperature, 30 min; (iv) TFA, CH2Cl2, room temperature, 10 min; (v) pivaloyl
chloride/CH2Cl2, Et3N, from 0 C to room temperature, 30 min, then ReNH2, from 0 C to room temperature, 1e2 h; (vi) pivaloyl chloride/CH2Cl2, Et3N, from 0 C to room temperature, 30 min, then R0 -Aniline, from 0 C to room temperature, 2e7 h.
Scheme 3. Introduction of an oxygen-based group on position 6 of the 3-carboxamide
coumarin scaffold. Reagents and conditions: (i) aniline, microwave (180 C, 30 min);
(ii) N,N0 -diphenylmalonamide (30), piperidine, acetic acid, ethanol, reflux, 21 h.
we initially intent to use the structure of FXIIac (PDB: 4XE4) [35] for
docking experiments. However, Pathak et al. reported that their
structure displays an unexpected zymogen-like conformation [35].
Based on this consideration, we envisaged to build a homology
model of FXIIa. The homology model was built using the SWISSMODEL program [36,37] and was generated from the crystal
structure of the (active) hepatocyte growth factor activator (HGFAPDB: 2R0L; 43% similarity).
Then, we compared this homology model to the crystal structure (PDB: 4XE4) and our comparison was focused on the orientation of key amino acids. Our analysis (Supplementary material)
revealed that the homology model would be more suitable than the
currently available crystal structure for docking experiments but
several adjustments would be needed since key residues (Tyr99,
Arg73, Leu33, Leu59, Leu64, Leu106 and Trp35) are not properly
oriented in this model (Supplementary material). We thus hypothesized that the construction of a consensus model, that would
join the best features from the homology model and the crystal
structure, would be more relevant for docking studies. The building
of a consensus model, called “FXIIa hybrid model” in this publication, was inspired from the work of Bujnicki and coworkers [38]. To
achieve this, we superimposed the crystal structure and our homology model, suppressed the loop containing the flipping residues (Tyr99, Arg73, Leu33, Leu59, Leu64, Leu106 and Trp35) in the
homology model and replaced it by the loop from the crystal
structure (Fig. 2).
C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
185
Table 1
PhysChem score calculation and inhibition percentage on FXIIa for Series A.
Compound
Log pa
MW corra,b
PSAa,c (A2)
Solub. pH 6.5a,d (mg/mL)
Rot. Bondsa,e
Phys-Chem score
FXIIa Inhibition (%)f
X,Y
R
3
4
6-CH2NH2
6-
C6H5
C6H5
1.6
3.2
294.3
362.42
81.42
58.64
9.9
0.7
4
4
0
1
26 ± 0.5
28 ± 2.1
5
6-
C6H5
1.7
364.39
67.87
0.048
4
1
18 ± 1.0
6
6-
C6H5
2.7
348.4
58.64
3.4
4
0
21 ± 1.5
7
16
6-CH2N-(CH2eCH3)2
6-C1, 8-Br
C6H5
3.0
1.5
350.41
275.68
58.64
81.42
2.9
61.9
6
4
1
0
14 ± 2.4
23 ± 1.7
17
6-C1, 8-Br
1.8
345.77
67.87
0.4
4
0
17 ± 1.3
18
6,8-Cl
2.4
338.82
67.87
0.4
4
0
20 ± 2.2
19
6,8-Br
1.5
352.72
67.87
0.5
4
0
14 ± 3.3
20
6-Cl, 8-Br
1.8
303.73
58.64
2.4
4
0
26 ± 3.8
21
6,8-Cl
2.2
296.78
58.64
2.2
4
0
29 ± 1.0
22
6,8-Br
2.5
310.68
58.64
3
4
0
30 ± 2.5
1
6-C1, 8-Br
3.6
308.7
55.4
0.00059
2
3
90 ± 3.3
a
b
c
d
e
f
C6H5
Property predicted with the ACD/Labs software (module Phys-Chem predictions e version 12.01).
MWcorr ¼ molecular weight corrected for halogens.
PSA ¼ polar Surface Area.
Solub. pH 6.5 ¼ solubility at pH 6.5.
Rot. Bonds ¼ rotable bonds.
Results are expressed in mean ± SD.
In the FXIIa hybrid model, residues are thus set in their putative
active orientation (Supplementary material). Also, the active site
pockets are of particular interest since they interact with inhibitors.
First, the so-called specificity pocket S1, which is mainly characterized by Asp189 and Tyr228, is conserved in all the trypsin-like
serine proteases. In their work, Emsley and coworkers [35] highlighted the presence of another pocket called S3/4 pocket. In this
pocket, Trp215 is a constant feature that is found in all the coagulation proteases while Tyr99 is present in a similar orientation in
FXII, t-PA, u-PA as well as in the more distant FXa [35]. Met180 is
also a constant feature in these proteases (Fig. 3). According to
Pathak et al., FXIIa is also characterized by a distinctive H1 pocket
located in front of the S1 pocket. The side chains of Leu33, Leu64,
Leu59, Leu106 and Trp35 contribute to the highly hydrophobic
character of this particular pocket (Fig. 3).
As a final step, the quality of the model was checked with the
VERIFY3D tool [38e41]. As stated by the wed-based server (http://
services.mbi.ucla.edu/Verify_3D/), this method analyzes the
compatibility of an atomic model (3D) with its own amino acid
sequence (1D). Each residue is assigned a structural class based on
its location and environment (alpha, beta, loop, polar, apolar, etc).
Then a database generated from good structures is used to obtain a
score for each of the 20 amino acids in this structural class. The
scores range from 1 to þ1 and the model passes the test when at
least 80% of the amino acids scored 0.2 in the 3D/1D profile
(http://services.mbi.ucla.edu/Verify_3D/). In our study, the hybrid
model passed the test since 96.5% of the amino acids scored 0.2.
2.4.2. Docking study
To obtain information about target-inhibitor interactions that
would help us to understand the structural requirements for FXIIa
inhibition, we performed a docking study using our hybrid model.
The docking simulations were carried out with all assessed compounds (1, 3e7, 16e28, 32) using the automated GOLD program
[42]. For each ligand, twenty solutions were generated and ranked
according to the ChemPLP scoring function. For each compound,
the best pose was retained.
First, the ability of our model to discriminate between active and
non-active compounds was evaluated. Interestingly, we observed
that active derivatives (23e25 and 27) have the 6,8-disubstituents
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Table 2
PhysChem score calculation and inhibition percentage on FXIIa for Series B.
Compound
Log Pa
MW corra,b (g/mol)
PSAa,c (A2)
Solub. pH 6.5a,d (mg/mL)
Rot. Bondsa,e
Phys-Chem score
FXIIa inhibition (%)f
X,Y
R
23
24
25
26
27
28
32
6-C1, 8-Br
6-C1, 8-Br
6-C1, 8-Br
6-C1, 8-Br
6-C1, 8-Br
6-C1, 8-Br
6-CH2OH
20 -OH, 50 -Cl
20 -OH, 40 -Cl
40 -OH
30 -OH
20 -OH
20 -COOH, 40 -Cl
eH
4.9
4.8
3.0
3.0
3.9
5.1
1.3
342.95
342.95
324.7
324.7
324.7
370.96
295.29
75.63
75.63
75.63
75.63
75.63
92.7
75.63
0.0001
0.00011
0.0015
0.0014
0.00071
0.036
0.052
3
3
3
3
3
3
4
3
3
3
3
3
3
0
99
99
90
90
99
75
14
1
6-C1, 8-Br
C6H5
3.6
308.7
55.4
0.00059
2
3
90 ± 3.3
a
b
c
d
e
f
Compound
1
23
24
25
26
27
a
c
0.8
4.1
3.0
0.7
1.1
10.6
0.4
Property predicted with the ACD/Labs software (module Phys-Chem predictions e version 12.01).
MWcorr ¼ molecular weight corrected for halogens.
PSA ¼ polar Surface Area.
Solub. pH 6.5 ¼ solubility at pH 6.5.
Rot. Bonds ¼ rotable bonds.
Results are expressed in mean ± SD.
Table 3
Potency and selectivity of promising compounds.
b
±
±
±
±
±
±
±
FXIIa IC50 (mM)a
b
16 (9e26)
5 (3e8)
8 (5e12)
41 (17e101)
34 (8e142)
7 (4e12)
Thrombin
c
NA
NA
NA
NA
NA
NA
Plasma kallikrein
TF/FVIIa
FXa
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
IC50 are expressed in mean and 95% confidence intervals.
In literature [25] 4.4 (3.2e6.2) mM in 10% DMSO instead of 5% in this study.
NA ¼ not active: inhibition <10% at 100 mM.
on the coumarin ring pointing towards the Trp35 residue from the
H1 pocket (Figs. 4A and 5B) whereas none of the inactive compounds displayed such orientation (Fig. 4B). For the active compounds 1 and 26, the best docking poses proposed a binding mode
where the coumarin ring was located in the S3/4 pocket and the 3substituent in the S1 pocket. However, poses from the second
cluster (60% and 50% of occurrence for 1 and 26, respectively) oriented the 6,8-halogen of the coumarin towards the H1 pocket
which was in agreement with the poses of the active compounds
23e25 and 27. Taken together, these results indicate that the model
may be considered as discriminating between active and inactive
compounds within the series presented in this study.
In the second step of our docking study, we aimed to investigate
the interactions between the active compounds and the enzyme in
Fig. 2. Building of the FXIIa hybrid model. In this model, we replaced the loop containing the flipping residues from the homology model by the loop from the crystal structure.
C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
Fig. 3. Surface structure of the FXIIa hybrid model. In the S1 pocket, Tyr228 is located
at the bottom. The S3/4 pocket described by Pathak et al. [35] is mainly delimited by
Tyr99, Trp215 and Met180. The hydrophobic pocket, called the H1 pocket by Emsley
and coworkers [35], is drawn by the side chains of Leu33, Leu59, Leu64, Leu106 (not
shown) and Trp35.
order to better explore the chemical features needed for the inhibition. To achieve this, we first minimized the ligands in the active
site and calculated the binding energies before visualizing the interactions with the Discovery Studio 4.0 (Accelrys Inç San Diego,
187
California, USA) program. The calculation of the binding energies
showed that the most active compound (23) has the lowest binding
energy (Supplementary material) suggesting that this compound is
better stabilized in the active site. On this basis, we decided to
further analyze the interactions between this compound and the
enzyme.
As depicted in Fig. 5A, the halogens from compound 23 are
pointing to Trp35 from the hydrophobic H1 pocket. Indeed, a palkyl interaction (Fig. 5B) was highlighted between the bromine
group in position 8 of the coumarin and the indole of Trp35 (8Br/Trp35, distance ¼ 4.69 Å). This bromine atom was also
involved in two other hydrophobic interactions with residues
Phe41 (8-Br/Phe41, distance ¼ 3.17 Å) and Cys42 (8-Br/Cys42,
distance ¼ 4.54 Å). The coumarin ring also interacted with the
enzyme thanks to the lactone that formed hydrogen bonds (COlactone/NHGly193, distance ¼ 1.92 Å, and COlactone/CHGln192,
distance ¼ 2.76 Å). The coumarin side chain was also engaged in the
establishment of interactions. Indeed, hydrogen bonds were found
between the hydroxyl group and Ser195 (20 -OH/OHSer195,
distance ¼ 1.84 Å) or the backbone of Cys220 (20 -OH/COCys220,
distance ¼ 1.90 Å). The phenyl and its chlorine atom both interact
with Cys220 thanks to p-alkyl (Phenyl/Cys220, distance ¼ 5.31 Å)
and alkyl interactions (50 -Cl/Cys220, distance ¼ 4.46 Å).
Regarding the stabilization in pockets, compound 23 seems to
interact at the entry of the S1 (through interactions with the
disulfure bridge Cys191-Cys220) and H1 (through interactions with
Trp35) pockets. The fact that the molecule does not fully fill these
Fig. 4. Poses of active compounds (A) and inactive (B) compounds in the active site of the hybrid model. (A) Active compounds have the 6,8-substituents of the coumarin ring
pointing to the H1 pocket. (B) None of the inactive compounds have their 6,8-halogens on the coumarin scaffold directed towards the H1 pocket.
Fig. 5. Compound 23 stabilized FXIIa active site (A) and details of interactions (B).
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C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
pockets could explain its micromolar range activity. Besides, the H1
pocket is a distinctive feature of FXIIa which could account for the
selectivity of compound 23.
Finally, we decided to rationalize this selectivity hypothesis in
silico. To achieve this, we superimposed the FXIIa hybrid modelcompound 23 complex with the crystal structures of thrombin
(PDB: 1TA2 [43]), FXa (PDB: 2W26 [44]), FVIIa (PDB: 4YT7 [45]) and
plasma kallikrein (PDB: 4OGX [46]).
The active site of thrombin gathers three pockets, namely the S,
D and P pockets (Fig. 6), that can be targeted in order to develop
inhibitors [24,43,47]. When superimposing FXIIa hybrid modelcompound 23 complex with thrombin (Fig. 6), we noticed a clash
of the coumarin ring. This clash occurred in the area of the H1
pocket in FXIIa meaning that such binding in thrombin is impossible. The H1 pocket in FXIIa is represented by Leu33, Leu59, Leu64,
Leu106 and Trp35. In thrombin, we found Leu33, Leu59, Leu64,
Met106 and Arg35 but these residues did not contribute to a
pocket.
The active site of FXa is restricted to the S1 and S4 pockets which
implies the design of L-shaped inhibitors [44]. Consequently, a
clash was observed when we superimposed compound 23 docked
in FXIIa with FXa (Fig. 7). Also, as in thrombin, residues Leu33,
Leu59, Phe64, Leu106 and Asn35 did not draw a (unreachable)
pocket (Fig. 7).
Coagulation FVIIa has 5 subsites (S1eS4 and S10 ) in its active site
[48]. The superimposition of our FXIIa hybrid model-compound 23
complex with FVIIa crystal structure (PDB: 4YT7) showed only a
small clash in the S1 pocket (Fig. 8). This may be not significant
since the size and shape of the FVIIa S1 pocket is very similar to the
S1 pocket in thrombin, FXa and trypsin [48]. As in thrombin and
FXa, residues Leu33, Phe59, Leu64, Leu106 and Val35 in FVIIa did
not contribute to a pocket. However, FVIIa possesses a hydrophobic
S10 pocket (with Cys42-Cys58 disulfide bridge, His57 and Leu41
delimiting the pocket) that could interact with a phenyl moiety
[45]. Considering the orientation of compound 23 due to the superimposition, it would be likely that the coumarin ring interacts
with this pocket. However, to effectively inhibit FVIIa, inhibitors
bind at least 3 subsites of the active site [48,49]. This fact might
explain why this derivative did not show an activity at 50 mM on
this protease.
Fig. 7. Superimposition of FXIIa hybrid model-compound 23 complex with FXa. In the
picture, FXIIa is hidden and only compound 23 is shown for clarity purpose. Active site
of FXa is restricted to S1 (Asp189) and S4 (Tyr99, Trp215, Phe174) pockets and residues
Leu33, Leu59, Phe64, Leu106 and Asn35 do not draw an unreachable pocket. This
caused a clashed of compound 23 after superimposition.
Fig. 8. Superimposition of FXIIa hybrid model-compound 23 complex with FVIIa. In
the picture, FXIIa is hidden and only compound 23 is shown for clarity purpose. FVIIa
has a S1 (Asp189), S2/4 (Trh99, Trp215, His57) and a S10 pocket (Cys42-Cys58, His57,
Leu41). Residues Leu33, Phe59, Leu64, Leu106 and Val35 do not participate in the
formation of a pocket.
Fig. 6. Superimposition of FXIIa hybrid model-compound 23 complex with thrombin.
In the picture, FXIIa is hidden and only compound 23 is shown for clarity purpose.
Thrombin pockets are the S (Asp189), D (Trp215, Ile174, Leu99) and P (Trp60D, Tyr60A)
pockets. The orientation adopted by compound 23 due to the superimposition lead to a
clash in the structure. Besides, we previously mentioned that residues Leu33, Leu59,
Leu64, Leu106 and Trp35 in FXIIa contribute to the H1 pocket but in thrombin Leu33,
Leu59, Leu64, Met106 and Arg35 are not involved in a pocket.
Fig. 9 illustrates the superposition of FXIIa hybrid modelcompound 23 complex with plasma kallikrein. This protease displays S1, S2, S3 and S10 pockets in its active site [46,50]. The
orientation of compound 23 given by the superimposition shows
that the molecule could interact with S1 and S10 pockets. This latter
involves the side chains of Leu41, Leu60B and Trp65D, as well as the
backbone and side chain of Asp60. This pocket is located in the
same area than the H1 pocket in FXIIa but is different in size and
shape. Therefore, it might be possible that compound 23 inhibits
plasma kallikrein but its binding may not be enough to display an
activity at 50 mM. Here also, residues 33, 59, 64, 106 and 35
(meaning Leu33, Phe59, Trp65D, Ile 106 and Val35) do not draw a
pocket.
In summary for this in silico selectivity part, we found that
residues contributing to the H1 pocket in FXIIa are different in
nature and they do not participate in the formation of a pocket in
the assessed proteases (thrombin, FXa, FVIIa and plasma kallikrein).
Among these proteases, only the S10 pocket of plasma kallikrein
C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
Fig. 9. Superimposition of FXIIa hybrid model-compound 23 complex with plasma
kallikrein. In the picture, FXIIa is hidden and only compound 23 is shown for clarity
purpose. Plasma kallikrein pockets are S1 (Asp189), S2 (Asp102), S3 (Trp215, Tyr174,
Gly99) and S10 (Leu41, Leu60B and Trp65D, Asp60) pockets. Residues Leu33, Phe59,
Trp65D, Ile 106 and Val35 do not contribute to a pocket but the S10 pocket looks like
the H1 pocket of FXIIa. However, the pockets are different in shape and size. Besides,
taking into account the orientation of compound 23 in the protease suggests that
interactions with the S1'pocket may be not excluded.
looks like the FXIIa H1 pocket but involves different residues
leading to a difference in shape and size. These data thus suggest
that selectivity could be obtained by targeting the H1 pocket of
FXIIa.
3. Conclusion
The discovery and development of FXIIa inhibitors is of valuable
interest for blood coagulation assays as well as for clinical applications implying the exposure of patients to artificial surfaces. In
this publication, we have developed 3-carboxamide coumarins
modulated in position 3 or 6 with amino- or oxygen-based group.
These modulations were introduced in the perspective to afford
selective FXIIa inhibitors with a better physico-chemical profile.
Among our series, the modulation of the carboxamide side chain
with an oxygen-based group afforded six active compounds with
IC50 in the micromolar range. To better understand the structureactivity relationship of our compounds, a docking study was performed. To this end, we generated a hybrid model of FXIIa. This
hybrid model was built from the published crystal structure of FXIIa
in its zymogen form and a homology model based on the X-ray
structure of the HGFA. Both initial 3D structures had limitations
rendering docking investigations unsuitable. Hence, the creation of
a hybrid model was a means to keep the advantages while overcoming the limitations of each structure. Interestingly, the molecular modeling study showed that the hybrid model seems to
discriminate active from inactive compounds within the series
described in this publication. Furthermore, the interactions highlighted for our most active compound helped to understand its
activity and selectivity.
4. Experimental section
4.1. Chemistry
Chemical reagents and solvents were used as received from
commercial sources (mainly Sigma-Aldrich. Acros and Fisher Scientific). Diethyl malonate (29) was purchased from Acros. The
syntheses of 2 [29], 9 [25], 10 [25] and 31 [22] were previously
described. The microwave-assisted syntheses were carried out in
189
an Initiator 16 single-mode microwave instrument producing
controlled irradiation at 2.450 GHz (BiotageAB, Uppsala, Sweden).
Reaction times refer to hold times at the temperatures indicated,
not to total irradiation times. The temperature was measured with
an IR sensor on the outside of the reaction vessel. 1H NMR spectra
were recorded in a DMSO-d6 or CDCl3 solution on a Jeol JNM EX 400
spectrometer at 400 MHz using tetramethylsilane (TMS) as internal
standard (d: 0.00 ppm). 13C spectra were recorded for tested compounds on the same spectrometer at 100 MHz. Chemical shifts (d)
are expressed in ppm downfield from tetramethylsilane. Melting
points were measured with a Büchi B-540 capillary melting point
apparatus in open capillaries and are uncorrected. Elemental analyses (C, H, N) for the newly synthesized 3-carboxamide coumarins
were performed on a Thermo Finnigan-FlashEA 1112 apparatus.
Automated flash chromatography was performed on a Biotage AB
SP1 system equipped with prepacked silica cartridges (GraceResolv™ 24 g. Grace. USA). Analytical LC/MS analyses were carried
out on an Agilent 1100 series HPLC coupled with a MSD Trap SL
system using UV detection at 254 and 361 nm. Mass spectra were
recorded using electron spray ionization operating in positive mode
(unless stated otherwise). An LC/MS analytical run consisted in the
injection of 10 ml of a 20 mg/mL acetonitrile solution onto a C18
3.5 mm Zorbax SB column (100 mm 3 mm). A gradient (flow rate
of 0.5 mL/min) of acetonitrile in acetic acid 0.1% (v/v in water) from
5% to 95% in acetonitrile over 5 min, holding for 3 min, then
reversing to 5% acetonitrile within 0.1 min and holding for an
additional 5.4 min was applied to allow the separation of compounds. All new molecules were determined to be >95% pure by
LC-UV.
4.1.1. General synthetic procedures
4.1.1.1. General procedure A for synthesis of compounds 4e7. A microwave vial (20 mL) equipped with a magnetic stirrer was loaded
with compound 2 (300 mg; 1 eq.) dissolved in 15 mL of ethanol and
the amine (2.5 eq.). The process vial was sealed and heated at
100 C for 10 min. After completion of the irradiation time, the
mixture was cooled (2e8 C) overnight. The precipitate formed was
filtered, then washed with ethanol and dried at 40 C under
vacuum.
4.1.1.2. General procedure B for synthesis of compounds 11e13.
A round-bottomed flask equipped with a magnetic stirrer bead was
filled with the appropriate 6,8-halogeno-2-oxo-2H-1-benzopyran3-carboxylic acid ethyl ester (1 g; 1 eq.) and sodium hydroxide 10%
(w/v in water) (10 mL; 8.3 eq.) [28]. After stirring at reflux temperature for 30 min, the mixture was cooled to room temperature
and 6 M hydrochloric acid (40.0 mL; 80 eq.) was slowly added. The
solid formed was filtered, washed with 6 M hydrochloric acid and
dried at 40 C under vacuum.
4.1.1.3. General procedure C for synthesis of compounds 17e22.
Each flask from a carousel (Radleys, Essex, UK) under an argon atmosphere
was
charged
with
6,8-halogeno-2-oxo-2H-1benzopyran-3-carboxylic acid (100e200 mg; 1 eq.) and dry
dichlorometane (10 mL). After cooling the mixture at 4 C, pivaloyl
chloride (1.1 eq.) and triethylamine (1 eq.) were successively added.
The solution was then stirred for 30 min at room temperature.
Upon completion of the reaction time, the mixture was cooled at
4 C and the amine (1.1 eq.) was added. The resulting mixture was
stirred for 1e2 h at room temperature. Upon completion of the
reaction time, the mixture was extracted twice with NaHCO3
saturated and water successively. The combined organic layers
were dried over MgSO4 and evaporated to dryness. The residue was
washed in boiling acetonitrile and filtered off. The filtrate was
cooled (2e8 C) overnight. The white precipitate formed was
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C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
filtered off, then washed with ethanol and dried at 40 C under
vacuum.
4.1.1.4. General procedure D for synthesis of compounds 23e28.
A round-bottomed flask under an argon atmosphere was charged
with compound 13 (100e400 mg; 1 eq.) and dry dichlorometane
(15 mL). After cooling the mixture at 4 C, pivaloyl chloride (1.1 eq.)
and triethylamine (1 eq.) were successively added. The solution was
then stirred for 30 min at room temperature. In the meantime, the
amine (1.1 eq.) is suspended in dry dichloromethane (10 mL) in a
doubled-neck flask cooled at 4 C and placed under an argon atmosphere. When the first reaction was completed, it was dropped
on the amine suspension over 15 min with a dropping funnel. The
resulting suspension was stirred for 2e7 h at room temperature.
TCL, workup and purification depended on the synthesized
compound.
4.1.1.4.1. 6-(Aminomethyl)-N-phenyl-2-oxo-2H-1-benzopyran-3carboxamide-chlorhydrate (3). A mixture of compound 2 (100 mg;
0.319 mmol) and hexamethylenetetramine (90 mg. 0.642 mmol) in
chloroform (15 mL) was stirred at 70 C for 18 h. The solvent was
then evaporated under reduced pressure to give the crude Delepine
adduct. Ethanol (5 mL), 6 M hydrochloric acid (1.350 mL) and water
(1.250 mL) were added to this crude mixture. After stirring at 90 C
for 90 min, the heterogeneous reaction mixture was cooled in an ice
bath and filtered to remove ammonium chloride (NH4Cl). The filter
was washed with ethanol and the filtrate was placed at 2e8 C
overnight. The yellow solid formed was filtered, then washed with
ethanol and dried at 40 C under vacuum. Yield: 49%. Mp
282.2e283.4 C. LC/MS tr ¼ 5.2 min, m/z [MH]þ ¼ 294.9. 1H NMR
(400 MHz, DMSO-d6) d 10.63 (s, 1H, NH), 8.79 (s, 1H, H-4), 8.52 (s,
3H, NHþ
3 ), 8.03 (d, J ¼ 1.8 Hz, 1H, H-7), 7.95e7.83 (m, 1H, H-5), 7.70
(d, J ¼ 7.8 Hz, 2H, H-20 , H-60 ), 7.60 (d, J ¼ 8.6 Hz, 1H, H-8), 7.36 (t,
J ¼ 7.9 Hz, 2H, H-30 , H-50 ), 7.12 (t, J ¼ 7.4 Hz, 1H, H-40 ), 4.08 (s, 2H,
CH2). 13C NMR (100 MHz, DMSO-d6): d ¼ 171.0, 160.7, 160.4, 154.2,
147.0, 138.5, 131.7, 131.3, 129.6, 129.6, 124.9, 121.4, 120.4, 120.4, 118.7,
117.2, 41.8. Anal. Calcd. for C17H14N2O3$HCl: C,61.73; H, 4.57; N, 8.47.
Found: C, 61.79; H, 4.66; N, 8.35.
4.1.1.4.2. N-Phenyl-6-(N-methylpiperidine)-2-oxo-2H-1benzopyran-3-carboxamide (4). 4 was prepared according to the
general procedure A from piperidine (0.239 mL; 2.40 mmol). The
title compound was obtained as a yellow powder. Yield: 55%. Mp
167.5e168.0 C. LC/MS tr ¼ 5.9, min m/z [MH]þ ¼ 363.0. 1H NMR
(DMSO-d6) d 10.64 (s, 1H, NH), 8.86 (s, 1H, H-4), 7.88e7.83 (m, 1H,
H-5). 7.71e7.62 (m, 3H, H-20 , H-60 , H-7), 7.47 (d, J ¼ 8.5 Hz, 1H, H-8),
7.39e7.31 (m, 2H, H-30 , H-50 ), 7.15e7.07 (m, 1H, H40 ), 3,45 (s, 2H,
CH2), 2.35e2.23 (m, 4H, CH2-20 , CH2-60 ), 1.50e1.34 (m, 6H, CH2-30 ,
CH2-40 , CH2-50 ). 13C NMR (100 MHz, DMSO-d6): d ¼ 153.5, 147.9,
136.5, 135.4, 134.0, 130.4, 129.6, 129.6, 126.4, 124.9, 120.4, 120.4,
120.4, 118.7, 116.5, 62.2, 54.4, 54.4, 26.1, 26.1, 24.4, 19.2. Anal. Calcd.
for C22H22N2O3: C,72.91; H, 6.12; N,7.73. Found: C,73.05; H,5.99;
N,7.78.
4.1.1.4.3. 6-(N-Methylmorpholine)-N-phenyl-2-oxo-2H-1benzopyran-3-carboxamide (5). 5 was prepared according to the
general procedure A from morpholine (0.212 mL; 2.40 mmol). The
solid obtained was purified by automated flash chromatography.
The elution started with an ethyl acetate/cyclohexane ratio of 12/88
over one column volume (CV); the ratio increased to 62/38 over 5
CV kept over 1 CV then increased to 100/0 over 7 CV and finally kept
at this ratio over 13.5 CV. The collection was monitored by UV
detection at 254 and 320 nm. The product was precipitated (yellow
solid) by evaporation of the solvents from the pooled column
fractions. Yield: 47%. Mp 199.4e199.9 C. LC/MS tr ¼ 5.5 min, m/z
[MH]þ ¼ 365.0. 1H NMR (CDCl3) d 10.84 (s, 1H, NH), 9.00 (s, 1H, H-4),
7.78e7.64 (m, 4H, H-20 , H-30 , H-50 , H-60 ), 7.43e7.32 (m, 3H, H-5, H-7,
H-8), 7.18e7.10 (m, 1H, H-40 ), 3.75e3.66 (m, 4H, CH2-30 , CH2-50 ),
3.58 (s, 2H, CH2), 2.47 (s, 4H, CH2-20 , CH2-60 ). 13C NMR (100 MHz,
DMSO-d6): d ¼ 161.0, 160.4, 153.6, 147.9, 138.5, 135.7, 135.4, 130.6,
129.6, 129.6, 124.8, 120.6, 120.4, 120.4, 118.8, 116.7, 66.7, 66.7, 61.8,
53.6, 53.6. Anal. Calcd. for C21H20N2O4: C,69.22; H,5.53; N,7.69.
Found: C,69.60; H,5.56; N,7.40
4.1.1.4.4. N-Phenyl-6-(N-methylpyrrolidine)-2-oxo-2H-1benzopyran-3-carboxamide (6). 6 was prepared according to the
general procedure A from pyrrolidine (0.199 mL; 2.40 mmol). The
title compound was obtained as a yellow powder. Yield: 56%. Mp
182.0e182.6 C. LC/MS tr ¼ 5.6 min, m/z [MH]þ ¼ 349.0. 1H NMR
(DMSO-d6) d 10.64 (s, 1H, NH), 8.86 (s, 1H, H-4), 7.89 (m, 1H, H-5),
7.68 (t, J ¼ 8.3 Hz, 3H, H-7, H-20 , H-60 ), 7.47 (d, J ¼ 8.7 Hz, 1H, H-8),
7.36 (t, J ¼ 7.6 Hz, 2H, H-30 , H-50 ), 7.12 (t, J ¼ 7.3 Hz, 1H, H-40 ), 3.62
(s, 2H, CH2), 2.47-2.35 (m, 4H, CH2-20 , CH2-50 ), 1.67 (m, 4H, CH2-30 ,
CH2-40 ). 13C NMR (100 MHz, DMSO-d6): d ¼ 171.1, 164.5, 160.5,
153.4, 148.0, 138.5, 137.3, 130.1, 129.6, 129.6, 124.8, 120.4, 120.3,
120.3, 118.8, 116.6, 59.1, 54.0, 54.0, 23.7, 23.7. Anal. Calcd. for
C21H20N2O3: C,72.40; H,5.79; N,8.04. Found: C,72.62; H,5.70;
N,7.87.
4.1.1.4.5. 6-(Diethylaminomethyl)-N-phenyl-2-oxo-2H-1benzopyran-3-carboxamide (7). 7 was prepared according to the
general procedure A from diethylamine (0.252 mL; 2.40 mmol). As
no precipitate was formed after the overnight cooling, the product
was concentrated under reduced pressure. The solid formed was
filtered, washed with ethanol and dried at 40 C under vacuum.
Yield: 52%. Mp 118.8e119.5 C. LC/MS tr ¼ 5.7 min, m/z
[MH]þ ¼ 351.1. 1H NMR (CDCl3) d 10.85 (s, 1H, NH), 9.00 (s, 1H, H-4),
7.76-7.67 (m, 4H, H-20 , H-30 , H-50 , H-60 ), 7.42-7.34 (m, 3H, H-5, H-7,
H-8), 7.15 (t, J ¼ 7.4 Hz, 1H, H-40 ), 3.64 (s, 2H, CH2), 2.55 (q, J ¼ 7.1 Hz,
4H, (CH2)2), 1.05 (t, J ¼ 7.1 Hz, 6H, (CH3)2). 13C NMR (100 MHz,
DMSO-d6): d ¼ 161.1, 160.5, 147.9, 138.5, 135.1, 130.0, 129.6, 129.6,
129.6, 124.8, 120.4, 120.3, 120.3, 118.7, 116.6, 116.6, 56.4, 46.6, 46.6,
12.2, 12.2. Anal. Calcd. for C21H22N2O3: C,71.98; H,6.33; N,7.99.
Found: C,71.11; H,6.33; N,7.67.
4.1.1.4.6. 6, 8-Dibromo-2-oxo-2H-1-benzopyran-3-carboxylic acid
ethyl ester (8). We followed the procedure described by Chimenti
[28] except for the workup. After completion of the reaction time,
boiling water (10 ml) was added to the brown-orange suspension.
The resulting yellow suspension is immediately cooled in an ice
bath to induce crystallization. The precipitate is filtered off, washed
twice with a mixture of methanol/water (60/40) and dried at 40 C
under vacuum. The title compound was obtained as a white powder. Yield: 76%. Mp 178.6e179.1 C. LC/MS tr ¼ 7.0 min, m/z
[MH]þ ¼ 374.9. 1H NMR (CDCl3) d 8.37 (s, 1H, H-4), 7.99-7.96 (m, 1H,
H-7), 7.71-7.67 (m, 1H, H-5), 4.48-4.36 (m, 2H, CH2), 1.45-1.15 (m,
3H, CH3).
4.1.1.4.7. 6, 8-Dibromo-2-oxo-2H-1-benzopyran-3-carboxylic acid
(11). 11 was prepared according to the general procedure B from
compound 8 (1.204 g; 3.20 mmol). Yield: 90%. Mp 225.2e225.5 C.
LC/MS tr ¼ 5.6 min, m/z [MH]þ ¼ 347.3. 1H NMR (DMSO-d6) d 13.49
(s, 1H, COOH), 8.65 (s, 1H, H-4), 8.22 (d, J ¼ 2.3 Hz, 1H, H-7), 8.15 (d,
J ¼ 2.3 Hz, 1H, H-5).
4.1.1.4.8. 6, 8-Dichloro-2-oxo-2H-1-benzopyran-3-carboxylic acid
(12). 12 was prepared according to the general procedure B from
compound 9 (1.002 g; 3.49 mmol). After filtration, the solid was
crystallized in acetonitrile to obtain the title compound. Yield: 50%.
Mp 203.1e204.1 C. LC/MS tr ¼ 5.4 min, m/z [MH]þ ¼ 259.0. 1H NMR
(CDCl3) d 11.91 (s, 1H, COOH), 8.85 (s, 1H, H-4), 7.81 (m, 1H, H-7),
7.65 (m, 1H, H-5).
4.1.1.4.9. 8-Bromo-6-chloro-2-oxo-2H-1-benzopyran-3carboxylic acid (13). 13 was prepared according to the general
procedure B from compound 10 (1.000 g; 3.02 mmol). Yield: 65%.
Mp 232.6e233.1 C. LC/MS tr ¼ 5.7 min, m/z [MH]þ ¼ 302.8. 1H NMR
(DMSO-d6) d 13.49 (s, 1H, COOH), 8.65 (s, 1H, H-4), 8.17-8.08 (m, 1H,
H-7), 8.07-7.97 (m, 1H, H-5).
C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
4.1.1.4.10. 8-Bromo-6-chloro-N-(2-(tert-butyl carbamate) ethyl)2-oxo-2H-1-benzopyran-3-carboxamide (15). Under an inert argon
atmosphere, a solution of the appropriate, compound 13 (447.3 mg;
1.47 mmol; 1 eq.) and thionyl chloride (1 mL/100 mg of initial
benzopyran) were stirred at reflux temperature for 3 h. The mixture
was cooled to room temperature and evaporated under reduced
pressure. The crude was dissolved in anhydrous toluene (1 mL/
100 mg of initial benzopyran) and evaporated. This dissolutionevaporation cycle was repeated three times. Then, the N-Boc-ethylenediamine (1.1 eq.) was added to the crude of acyl chloride (14)
dissolved in anhydrous dioxane (1 mL/100 mg of initial benzopyran). The resulting mixture was stirred at room temperature for
30 min. Upon completion of the reaction time, the solvent was
evaporated and the residue dissolved in chloromethane was
washed three times with 0.1 N hydrochloric acid. The combined
organic layers were dried over MgSO4 and evaporated to dryness.
The product was crystallized in acetonitrile. 15 was used for the
synthesis of compound 16 without purification. Yield: 21%. Mp
217e219 C. LC/MS tr ¼ 6.6 min, m/z [MH]þ ¼ 445.3. 1H NMR
(CDCl3) d 8.84 (s, 1H, NH), 8.78 (s, 1H, H-4), 7.86 (d, J ¼ 2.4 Hz, 1H, H7), 7.62 (d, J ¼ 2.3 Hz, 1H, H-5), 4.80 (s, 1H, NH), 3.58 (dd, J ¼ 12.0,
6.0 Hz, 2H, CH2), 3.37 (dd, J ¼ 14.0, 8.3 Hz, 2H, CH2), 1.43 (s, 9H,
(CH3)3).
4.1.1.4.11. 8-Bromo-6-chloro-N-ethylamine-2-oxo-2H-1benzopyran-3-carboxamide-trifluoroacetic acid salt (16). To a flask
containing 15 (65.8 mg; 0.190 mmol) under an argon atmosphere,
dry dichloromethane (3 mL) and trifluoroacetic acid (TFA - 1 mL)
were slowly added with a syringe. After stirring the pale pink solution at room temperature for 10 min, solvents were evaporated
under vacuum. The oily residue was suspended in acetonitrile and
the resulting mixture was kept at 2e8 C overnight. The white
precipitate formed was filtered, washed with acetonitrile and dried
at 40 C under vacuum. The filtrate was evaporated and acetonitrile
was added to the residue. The precipitate was filtered off, washed
with acetonitrile and dried at 40 C under vacuum. The two isolated
products were pooled. Presence of TFA was pointed out by
elemental analysis. Yield: 60%. Mp 236.7e237.6 C. LC/MS
tr ¼ 5.1 min, m/z [MH]þ ¼ 344.8. 1H NMR (DMSO-d6) d 8.84e8.77
(m, 2H, NH, H-4), 8.20e8.15 (m, 2H, H-5, H-7), 7.80 (s, 2H, NH2),
3.54 (q, J ¼ 6.0 Hz, 2H, CH2), 2.97 (t, J ¼ 6.1 Hz, 2H, CH2). 13C NMR
(100 MHz, DMSO-d6): d ¼ 161.9, 161.9, 159.5, 150.0, 146.9, 136.3,
129.7, 129.4, 121.2, 121.1, 110.4, 110.4, 38.9, 37.7. Anal. Calcd. for
C14H11BrClF3N2O5: C,36.59; H,2.41; N,6.10. Found: C,37.00; H,2.48;
N,5.76.
4.1.1.4.12. 8-Bromo-6-chloro-N-(2-morpholinoethyl)-2-oxo-2H1-benzopyran-3-carboxamide (17). 17 was prepared according to
the general procedure C from 13 (200.0 mg; 0.659 mmol) and 4-(2aminoethyl)-morpholine (95.2 mL; 0.725 mmol). The mixture was
stirred for 2 h after the addition of the amine. The two products
isolated from the first filtration and the crystallization were pooled.
Yield: 39%. Mp 208.9e209.4 C. LC/MS tR ¼ 5.5 min, m/z
[MH]þ ¼ 414.8. 1H NMR (CDCl3) d 9.06 (s, 1H, NH), 8.77 (s, 1H, H-4),
7.85 (d, J ¼ 2.3 Hz, 1H, H-7), 7.61 (d, J ¼ 2.3 Hz, 1H, H-5), 3.80e3.70
(m, 4H, CH2-30 , CH2-50 ), 3.61e3.53 (m, 2H, CH2), 2.64e2.45 (m, 6H,
CH2, CH2-20 , CH2-60 ). 13C NMR could not be obtained in NMR solvents due to precipitation issue. Anal. Calcd. for C16H16BrClN2O4:
C,46.23; H,3.88; N,6.74. Found: C,46.21; H,3.82; N,6.40.
4.1.1.4.13. 6, 8-Dichloro-N-(2-morpholinoethyl)-2-oxo-2H-1benzopyran-3-carboxamide (18). 18 was prepared according to the
general procedure C from 12 (200.0 mg; 0.772 mmol) and 4-(2aminoethyl)-morpholine (111.6 mL; 0.849 mmol). The yellow suspension was stirred for 1 h after the addition of the amine. Yield:
10%. Mp 210.9e211.4 C. LC/MS tr ¼ 5.3 min, m/z [MH]þ ¼ 370.9. 1H
NMR (CDCl3) d 9.06 (s, 1H, NH), 8.79 (s, 1H, H-4), 7.68 (d, J ¼ 2.3 Hz,
1H, H-7), 7.57 (d, J ¼ 2.3 Hz, 1H, H-5), 3.84e3.64 (m, 4H, CH2-30 ,
191
CH2-50 ), 3.57 (dd, J ¼ 11.5, 5.8 Hz, 2H, CH2), 2.70e2.39 (m, 6H, CH2,
CH2-20 , CH2-60 ). 13C NMR could not be obtained in NMR solvents
due to precipitation issue. Anal. Calcd. for C16H16Cl2N2O4: C,51.77;
H,4.34; N,7.55. Found: C,51.95; H,4.38; N,7.49.
4.1.1.4.14. 6, 8-Dibromo-N-(2-morpholinoethyl)-2-oxo-2H-1benzopyran-3-carboxamide (19). 19 was prepared according to the
general procedure C from 11 (130.0 mg; 0.374 mmol) and 4-(2aminoethyl)-morpholine (54 mL; 0.411 mmol). The yellow suspension was stirred for 1 h after the addition of the amine. Yield: 13%.
Mp 206.0e206.5 C. LC/MS tR ¼ 5.7 min, m/z [MH]þ ¼ 458.9. 1H
NMR (CDCl3) d 9.05 (s, 1H, NH), 8.77 (s, 1H, H-4), 7.99 (d, J ¼ 2.2 Hz,
1H, H-7), 7.76 (d, J ¼ 2.2 Hz, 1H, H-5), 3.84e3.61 (m, 4H, CH2-30 ,
CH2-50 ), 3.57 (dd, J ¼ 11.2, 5.6 Hz, 2H, CH2), 2.71e2.34 (m, 6H, CH2,
CH2-20 , CH2-60 ). 13C NMR could not be obtained in NMR solvents
due to precipitation issue. Anal. Calcd. for C16H16Br2N2O4: C,41.77;
H,3.50; N,6.09. Found: C,41.9; H,3.50; N,6.04.
4.1.1.4.15. 8-Bromo-6-chloro-N-(2-(dimethylamino)ethyl)-2-oxo2H-1-benzopyran-3-carboxamide (20). 20 was prepared according
to the general procedure C from 13 (300.0 mg; 0.989 mmol) and
dimethylethylenediamine (118.8 mL; 1.088 mmol). The suspension
was stirred for 1 h after the addition of the amine. Yield: 15%. Mp
207.4e208.4 C. LC/MS tr ¼ 5.4 min, m/z [MH]þ ¼ 372.8. 1H NMR
(CDCl3) d 8.91 (s, 1H, NH), 8.76 (s, 1H, H-4), 7.84 (d, J ¼ 2.4 Hz, 1H, H7), 7.61 (d, J ¼ 2.3 Hz, 1H, H-5), 3.55 (dd, J ¼ 11.7, 6.2 Hz, 2H, CH2),
2.53 (m, 2H, CH2), 1.43 (s, 6H, (CH3)2). 13C NMR (100 MHz, CDCl3):
d ¼ 160.6, 159.8, 149.8, 146.6, 136.7, 130.9, 128.0, 120.5, 120.4, 111.0,
57.8, 45.4, 45.4, 37.90. Anal. Calcd. for C14H14BrClN2O3: C,45.00;
H,3.78; N,7.50. Found: C,45.11; H,3.85; N,7.44.
4.1.1.4.16. 6, 8-Dichloro-N-(2-(dimethylamino)ethyl)-2-oxo-2H1-benzopyran-3-carboxamide (21). 21 was prepared according to
the general procedure C from 12 (300.0 mg; 1.158 mmol) and
dimethylethylenediamine (139.2 mL; 1.275 mmol). The suspension
was stirred for 1 h after the addition of the amine. Yield: 5%. Mp
205.1e205.6 C. LC/MS tr ¼ 5.3 min, m/z [MH]þ ¼ 328.9. 1H NMR
(CDCl3) d 8.90 (s, 1H, NH), 8.78 (s, 1H, H-4), 7.68 (d, J ¼ 2.4 Hz, 1H, H7), 7.56 (d, J ¼ 2.3 Hz, 1H, H-5), 3.55 (dd, J ¼ 11.7, 6.0 Hz, 2H, CH2),
2.53 (m, 2H, CH2), 2.29 (s, 6H, (CH3)2). 13C NMR (100 MHz, CDCl3):
d ¼ 160.7, 159.7, 148.7, 146.6, 133.7, 130.5, 127.3, 122.8, 120.6, 120.5,
57.8, 45.4, 45.4, 37.9. Anal. Calcd. for C14H14Cl2N2O3: C,51.08; H,4.29;
N,8.51. Found: C,50.97; H,4.31; N, 8.47.
4.1.1.4.17. 6, 8-Dibromo-N-(2-(dimethylamino)ethyl)-2-oxo-2H1-benzopyran-3-carboxamide (22). 22 was prepared according to
the general procedure C from 11 (120.0 mg; 0.345 mmol) and
dimethylethylenediamine (41.4 mL; 0.379 mmol). The suspension
was stirred for 1 h after the addition of the amine. Yield: 11%. Mp
216.2e217.2 C. LC/MS tr ¼ 5.5 min, m/z [MH]þ ¼ 416.8. 1H NMR
(CDCl3) d 8.91 (s, 1H, NH), 8.76 (s, 1H, H-4), 7.98 (d, J ¼ 2.2 Hz, 1H, H7), 7.75 (d, J ¼ 2.1 Hz, 1H, H-5), 3.55 (dd, J ¼ 11.2, 5.8 Hz, 2H, CH2),
2.61e2.44 (m, 2H, CH2), 2.31 (s, 6H, (CH3)2). 13C NMR could not be
obtained due to lack of material. Anal. Calcd. for C14H14Br2N2O3:
C,40.22; H,3.38; N,6.70. Found: C,39.93; H,3.28; N,6.51.
4.1.1.4.18. 8-Bromo-6-chloro-N-(50 -chloro-20 -hydroxyphenyl)-2oxo-2H-1-benzopyran-3-carboxamide (23). 23 was prepared according to the general procedure D from 13 (400.6 mg; 1.320 mmol)
and 2-hydroxy-5-chloro-aniline (208.5 mg; 1.452 mmol). The suspension was stirred for 1h30 after the addition on the amine. Upon
completion of the reaction time, the mixture is filtered off, washed
with ethyl acetate and dried at 40 C under vacuum. Yield: 79%. Mp
334.0e334.5 C. LC/MS tr ¼ 7.4 min, m/z [MH] - ¼ 425.6. 1H NMR
(DMSO-d6) d 11.04 (s, 1H, OH), 10.66 (s, 1H, NH), 8.96e8.93 (m, 1H,
H-4), 8.44e8.39 (m, 1H, H-5), 8.24e8.18 (m, 2H, H-40 , H-60 ),
7.04e6.96 (m, 1H, H-7), 6.91e6.85 (m, 1H, H-30 ). 13C NMR could not
be obtained in NMR solvents due to precipitation issue. Anal. Calcd.
for C16H8BrCl2NO4: C,44.79; H,1.88; N,3.26. Found: C,44.61; H,1.98;
N,3.22.
192
C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
4.1.1.4.19. 8-Bromo-6-chloro-N-(40 -chloro-20 -hydroxyphenyl)-2oxo-2H-1-benzopyran-3-carboxamide (24). 24 was prepared according to the general procedure D from 13 (307.7 mg; 1.014 mmol)
and 2-hydroxy-4-chloro-aniline (160.2 mg; 1.115 mmol). The suspension was stirred for 1h30 after the addition on the amine. Upon
completion of the reaction time, the mixture is filtered off, washed
with ethyl acetate and dried at 40 C under vacuum. Yield: 76%. Mp
357.6e358.6 C. LC/MS tr ¼ 7.4 min, m/z [MH] - ¼ 425.6. 1H NMR
(DMSO-d6) d 10.98 (s, 1H, NH), 10.90 (s, 1H, OH), 8.95 (s, 1H, H-4),
8.36 (d, J ¼ 8.4 Hz, 1H, H-60 ), 8.20 (m, 2H, H-5, H-7), 6.88 (d,
J ¼ 8.4 Hz, 2H, H-30 , H-50 ). 13C NMR could not be obtained in NMR
solvents due to precipitation issue. Anal. Calcd. for C16H8BrCl2NO4:
C,44.79; H,1.88; N,3.26. Found: C,44.74; H,1.90; N,3.14.
4.1.1.4.20. 8-Bromo-6-chloro-N-(40 -hydroxyphenyl)-2-oxo-2H-1benzopyran-3-carboxamide hydrated (25). 25 was prepared according to the general procedure D from 13 (202.8 mg;
0.668 mmol) and 4-hydroxy-aniline (80.2 mg; 0.735 mmol). The
suspension was stirred for 3 h after the addition on the amine. Upon
completion of the reaction time, the mixture is filtered off and
washed with ethyl acetate. As purification, the product was stirred
in boiling water for 30 min, filtered off, washed with water and
dried at 40 C under vacuum. Yield: 31%. Mp 293.4e294.3 C. LC/MS
tr ¼ 6.8 min, m/z [MH] - ¼ 391.7. 1H NMR (DMSO-d6) d 10.29 (s, 1H,
NH), 9.37 (s, 1H, OH), 8.77 (s, 1H, H-4), 8.15 (dd, J ¼ 17.5, 2.0 Hz, 2H,
H-5, H-7), 7.47 (d, J ¼ 8.6 Hz, 2H, H-20 , H-60 ), 6.74 (d, J ¼ 8.7 Hz, 2H,
H-30 , H-50 ). 13C NMR (100 MHz, DMSO-d6): d ¼ 159.8, 159.1, 154.8,
149.9, 145.8, 136.1, 129.9, 129.7, 129.2, 122.7, 122.2, 122.2, 121.4,
115.9, 115.9, 110.5. Anal. Calcd. for C16H9BrClNO4$H2O: C,46.57;
H,2.69; N,3.39. Found: C,46.83; H,2.38; N,3.21.
4.1.1.4.21. 8-Bromo-6-chloro-N-(30 -hydroxyphenyl)-2-oxo-2H-1benzopyran-3-carboxamide (26). 26 was prepared according to the
general procedure D from 13 (230.2 mg; 0.758 mmol) and 3hydroxy-aniline (91.1 mg; 0.834 mmol). The suspension was stirred for 2 h after the addition on the amine. Upon completion of the
reaction time, the mixture is filtered off and washed with
dichloromethane. As purification, the product was stirred in boiling
ethyl acetate for 10 min, filtered off, washed with ethyl acetate and
dried at 40 C under vacuum. Yield: 76%. Mp 312.2e313.0 C. LC/MS
tr ¼ 6.9 min, m/z [MH] - ¼ 391.6. 1H NMR (DMSO-d6) d 10.41 (s, 1H,
NH), 9.54 (s, 1H, OH), 8.76 (s, 1H, H-4), 8.16 (d, J ¼ 21.5 Hz, 2H, H-5,
H-7), 7.26 (s, 1H, H-20 ), 7.13 (t, J ¼ 8.1 Hz, 1H, H-60 ), 6.99 (d,
J ¼ 7.9 Hz, 1H, H-40 ), 6.52 (d, J ¼ 8.4 Hz, 1H, H-50 ). 13C NMR
(100 MHz, DMSO-d6): d ¼ 159.7, 158.4, 158.4, 149.9, 145.9, 139.3,
136.2, 130.3, 129.7, 129.2, 122.9, 121.3, 112.1, 111.1, 110.5, 107.4. Anal.
Calcd. for C16H9BrClNO4: C,48.70; H,2.30; N,3.55. Found: C,49.10;
H,2.26; N,3.62.
4.1.1.4.22. 8-Bromo-6-chloro-N-(20 -hydroxyphenyl)-2-oxo-2H-1benzopyran-3-carboxamide (27). 27 was prepared according to the
general procedure D from 13 (200.0 mg; 0.659 mmol) and 2hydroxy-aniline (79.1 mg; 0.725 mmol). The suspension was stirred for 2h30 after the addition on the amine. Upon completion of
the reaction time, the mixture was filtered off and washed with
ethyl acetate and dried at 40 C under vacuum. Yield: 58%. Mp
336.8e337.4 C. LC/MS tr ¼ 7.1 min, m/z [MH] - ¼ 391.6. 1H NMR
(DMSO-d6) d 10.96 (s, 1H, NH), 10.27 (s, 1H, OH), 8.95 (s, 1H, H-4),
8.35 (dd, J ¼ 8.0, 1.4 Hz, 1H, H-7), 8.22e8.16 (m, 2H, H-5, H-60 ),
6.97e6.76 (m, 3H, H-30 , H-40 , H-50 ). 13C NMR could not be obtained
in NMR solvents due to precipitation issue. Anal. Calcd. for
C16H9BrClNO4: C,48.70; H,2.30; N,3.55. Found: C,48.96; H,2.39;
N,3.74.
4.1.1.4.23. 8-Bromo-6-chloro-N-(20 -carboxylic
acid-40 -chlorophenyl)-2-oxo-2H-1-benzopyran-3-carboxamide (28). 28 was prepared according to the general procedure D from 13 (300.4 mg;
0.990 mmol) and 2-amino-5-chloro-benzoic acid (186.8 mg;
0.109 mmol). The suspension was stirred for 7 h after the addition
on the amine. Upon completion of the reaction time, the mixture is
filtered off and washed with ethyl acetate. As purification, the
product was stirred in 0.1 M hydrochloric acid for 10 min, filtered
off, washed with water and dried at 40 C under vacuum. Yield:
28%. Mp 321.3e321.9 C. LC/MS tr ¼ 7.3 min, m/z [MH] - ¼ 453.6.
1
H NMR (DMSO-d6) d 12.40 (s, 1H, OH), 8.93 (s, 1H, H-4), 8.69 (d,
J ¼ 9.0 Hz, 2H, H-30 , NH), 8.18 (s, 2H, H-5, H-7), 7.92 (d, J ¼ 7.9 Hz, 1H,
H-60 ), 7.70 (dd, J ¼ 9.0, 2.6 Hz, 1H, H-50 ). 13C NMR (100 MHz, DMSOd6): d ¼ 167.5, 160.2, 159.00, 150.2, 148.0, 138.7, 136.5, 133.5, 130.9,
129.6, 129.5, 127.9, 123.8, 121.6, 121.6, 121.3, 110.4. Anal. Calcd. for
C17H8BrCl2NO5: C,44.67; H,1.76; N,3.06. Found: C,44.53; H,1.87;
N,3.01.
4.1.1.4.24. N, N- diphenylmalonamide (30) [31]. A microwave
process vial (5 mL) equipped with a magnetic stirrer bead was
charged with compound 29 (814 mL; 5.362 mmol) and aniline
(1.96 mL; 21.45 mmol). The process vial was sealed and heated at
180 C for 30 min. After completion of the irradiation time, ethanol
was added to the mixture. The precipitate formed was filtered,
washed with ethanol and dried at 40 C under vacuum. Yield: 69%.
Mp 227.1e227.9 C. LC/MS tr ¼ 5.9 min, m/z [MH] - ¼ 252.8. 1H
NMR (DMSO-d6) d 10.15 (s, 2H, NH), 7.62e7.51 (m, 4H, H-2, H-6, H20 , H-60 ), 7.28 (t, J ¼ 7.8 Hz, 4H, H-3, H-5, H-30 , H-50 ), 7.02 (t,
J ¼ 7.1 Hz, 2H, H-4, H-40 ), 3.43 (s, 2H, CH2).
4 .1.1. 4 . 2 5 . 6 - ( H y d r o x y m e t hyl ) - N - p h e n yl - 2 - o x o - 2 H - 1 benzopyran-3-carboxamide (32). A round-bottomed flask was
charged with 30 (500 mg; 1.966 mmol), 31 [22] (598 mg;
3.932 mmol), piperidine (25 mL; 0.25 mmol), acetic acid (12.5 ml;
0.22 mmol) and ethanol (5 mL), and the resulting mixture was
stirred at reflux temperature for 21 h. After completion of the reaction time, the precipitate was filtered off, washed with ethanol
and dried at 40 C under vacuum. The title compound was obtained
as a light yellow powder. Yield: 90%. Mp 202.7e203.7 C. LC/MS
tr ¼ 6.1 min, m/z [MH]þ ¼ 296.0. 1H NMR (DMSO-d6) d 10.64 (s, 1H,
NH), 8.87 (s, 1H, H-4), 7.91e7.86 (m, 1H, H-5), 7.73e7.65 (m, 3H, H20 , H-60 , H-7), 7.49 (d, J ¼ 8.6 Hz, 1H, H-8), 7.36 (t, J ¼ 7.9 Hz, 2H, H-30 ,
H-50 ), 7.15e7.08 (m, 1H, H-40 ), 5.41 (m, 1H, OH), 4.55 (d, J ¼ 5.6 Hz,
2H, CH2). 13C NMR (100 MHz, DMSO-d6): d ¼ 161.1, 160.4, 153.4,
148.0, 140.3, 138.5, 133.2, 129.5, 129.5, 129.5, 128.0, 124.8, 120.4,
120.4, 118.6, 116.5, 62.4. Anal. Calcd. for C17H13NO4: C,69.15; H,4.44;
N,4.74. Found: C,69.06; H,4.43; N,4.66.
4.2. Enzymatic assays
4.2.1. Reagents
The activity of coumarins against human purified serine proteases was measured using chromogenic assays in 96-well microtiter plates. FXIIa, FXIa, FXa and plasma kallikrein were obtained
from Enzyme Research Laboratories (Swansea, UK), thrombin from
Roche Diagnostics (Mannheim, Germany), and FVIIa (NovoSeven®)
from Novo Nordisk. Chromogenic substrates S-2302, S-2366, S2765, S-2238 were purchased from Chromogenix Instrumentation
Laboratory (Bubendorf, Switzerland) and Chromozym t-PA from
Roche Diagnostics (Mannheim, Germany). For each assay, the optical density at 405 nm (OD405 nm) was measured in a microplate
reader. All coumarin compounds were prepared in DMSO. They
were assayed at 50 mM (in the final mixture) for the screening assay
on FXIIa and at 100 mM (in the final mixture) for the selectivity
study. For the IC50 determination of selected compounds on FXIIa,
nine concentrations ranging from 100 nM to 100 mM (in the final
mixture) were used and compounds were assayed in duplicate.
4.2.2. Procedures
4.2.2.1. FXIIa assay. 10 mL of test compound in vehicle (or vehicle
alone in the control samples) and 20 mL of human FXIIa (145 nM) in
150 mL of 0.5 mM Tris-Imidazole, 0.15 M NaCl buffer, pH 7.9 were
C. Bouckaert et al. / European Journal of Medicinal Chemistry 110 (2016) 181e194
incubated during 10 min in a 96-well assay plate at 25 C. 20 mL of
S-2302 (2.5 mM) were then added to initiate the enzymatic reaction. After 30 min of substrate hydrolysis, the reaction was stopped
by the addition of 20 mL acetic acid 10% (v/v in water) and the OD405
nm was measured. The background absorbance was read just before
adding the enzyme.
4.2.2.2. Thrombin assay. 20 mL of test compound in vehicle (or
vehicle alone in the control samples) and 20 mL human thrombin
(23.2 nM) in 140 mL of 0.01 M Tris-HCl, 0.01 M Hepes, 0.1 M NaCl,
0.1% (w/v) PEG 6000 buffer, pH 7.5 were incubated during 10 min in
a 96-well assay plate at 25 C. 20 mL of S-2238 (2.5 mM) were then
added to initiate the enzymatic reaction. The OD405 nm was
continuously monitored for 5 min. Then, the OD405 nm was plotted
against the time and the slope was calculated.
4.2.2.3. FXa assay. The same protocol as described for thrombin
was followed using human FXa (58 nM), a 0.05 M Tris-HCl, 0.005 M
CaCl2, 0.15 M NaCl, 0.001 M EDTA, 0.05% (v/v) Tween 20 buffer, pH
7.5 and the S-2765 substrate (1 mM).
4.2.2.4. Plasma kallikrein assay. The same protocol as described for
thrombin was followed using human plasma kallikrein (50 nM), a
0.05 M Tris-HCl, 0.5 mM EDTA, 0.05% (v/v) Tween 20, 0.15 M NaCl
buffer, pH 7.4 and the S-2302 substrate (4 mM).
4.2.2.5. TF/FVIIa assay. 20 mL of test compound in vehicle (or
vehicle alone in the control samples) and 20 mL human FVIIa
(50 nM) in 140 mL Innovin® dissolved with 0.1 M Tris-HCl, 0.3 M
NaCl, 0.01 M CaCl2, 0.1% (w/v) BSA buffer, pH 7.5 were incubated
during 10 min in a 96-well assay plate at 25 C. 20 mL of Chromozym
t-PA (5 mM) were then added to the mixture to initiate the enzymatic reaction. The OD405 nm was continuously monitored for
10 min. Then, the OD405 nm was plotted against the time and the
slope was calculated.
4.2.3. Inhibition percentage and IC50 determinations
The inhibition percentage on FXIIa was obtained by using the
following equation:
½1 ðDrug OD405 nm
Drug background OD405 nm =Vehicle OD405 nm
Vehicle background OD405 nm Þ*100
The IC50 on FXIIa were measured thanks to a HPLC-UV method.
This method aims at quantifying the para-nitroaniline (p-NA)
released during the enzymatic assay. The samples were prepared
by adding 100 mL of acetonitrile to 100 mL of a well medium. Each
well (i.e each replicate of molecules at several concentrations and
vehicle) used for the enzymatic assay is prepared for HPLC-UV
analysis.
The experiments were carried out on an Agilent 1100 series
HPLC. p-NA detection was made at 375 nm. During a run, 10 mL of
the sample solution was injected onto a C18 3.5 mm Zorbax SB
column (100 mm 3 mm). A gradient (flow rate of 0.5 mL/min) of
acetonitrile in acetic acid 0.1% (v/v in water) from 5% to 95% in
acetonitrile over 5 min, holding for 3 min, then reversing to 5%
acetonitrile within 0.1 min and holding for an additional 5.4 min
was applied to allow the separation. The AUC of the p-NA peak
(tr ¼ 5.2 min) for each assay was determined.
Using the HPLC-UV, the inhibition percentage was obtained by
using the following equation: [1-(AUCdrug/AUCvehicle)]*100. For
the IC50 determination of test compounds on FXIIa, a sigmoid doseresponse curve was drawn by plotting inhibition percentages
193
against the logarithm of the respective concentrations. The IC50
was then calculated with a non-linear regression equation using
GraphPad Prism, version 5.01 (GraphPad Software). The reported
IC50 are expressed as mean and 95% confidence intervals are in
parentheses.
For thrombin, FXa, plasma kallikrein and TF/FVIIa, the inhibition
percentage was obtained by using the following equation: [1-(slope
inhibitor curve/slope vehicle curve)]*100
4.3. Molecular modeling
Coumarins were sketched with Chem3D Pro 14.0 (Perkin Elmer
Informatics) and prepared for docking with Discovery Studio 4.0
(Accelrys Inç San Diego, California, USA). The hybrid model was also
prepared with this software. The docking experiments were carried
out with the automated GOLD 5.2.2 program [42]. The binding site
was defined as a 15 Å sphere from the oxygen of the hydroxyl group
of Ser195 (OgSer195). This sphere allowed taking into account the
residues from the S1, S3/4 and H1 pockets. For each ligand, the
number of genetic algorithm (GA) run was set at 100. We used the
default ChemPLP score function and the search efficiency was fixed
at 100%. For the output, we asked GOLD to keep the twenty best
solutions for each ligand. The in situ minimization of the actives
compounds (poses selected from the docking with GOLD), free
binding energy calculations and interactions visualization were
performed with Discovery Studio (DS) 4.0 (Accelrys Inç San Diego,
California, USA). The in situ minimization and free binding energy
calculation were launched in the same DS protocol. In this protocol,
the ligand conformational entropy [51,52] was also considered
(conformers generated with the BEST algorithm) and the Generalized Born with Molecular Volume (GBMV) was used as implicit
solvent model.
Conflict of interest
The authors have no conflict of interest to disclose.
Acknowledgments
C.B is a research fellow of the Fonds de la Recherche
Scientifique-FNRS and her grant supported this research. The authors thank Anne-Marie Murray and Christelle Vancraeynest for
their assistance. Part of this research used resources of the “Plateforme Technologique de Calcul Intensif (PTCI)” located at the University of Namur, Belgium, which is supported by the F.R.S.-FNRS
under the convention No. 2.4520.11. The PTCI is member of the
“Consortium des Equipements
de Calcul Intensif (CECI)”
(http://
www.ceci-hpc.be).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.01.023.
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