Novel bis-arylalkylamines as myeloperoxidase inhibitors: Design, synthesis, and structure-activity relationship study

Article abstract of DOI:10.1016/j.ejmech.2016.07.053

Human myeloperoxidase (MPO) plays an important role in innate immunity but also aggravates tissue damage by oxidation of biomolecules at sites of inflammation. As a result from a recent high-throughput virtual screening approach for MPO inhibitors, bis-2,2′-[(dihydro-1,3(2H,4H) pyrimidinediyl)bis(methylene)]phenol was detected as a promising lead compound for inhibition of the MPO-typical two-electron oxidation of chloride to hypochlorous acid (IC₅₀= 0.5 μM). In the present pharmacomodulation study, 37 derivatives of this lead compound were designed and synthesized driven by comprehensive docking studies and the impact on the chlorination activity of MPO. We describe the structural requirements for optimum (i) binding to the heme periphery and (ii) inhibition capacity. Finally, the best three inhibitors (bis-arylalkylamine derivatives) were probed for interaction with the MPO redox intermediates Compound I and Compound II. Determined apparent bimolecular rate constants together with determination of reduction potential and nucleophilicity of the selected compounds allowed us to propose a mechanism of inhibition. The best inhibitor was found to promote the accumulation of inactive form of MPO-Compound II and has IC₅₀= 54 nM, demonstrating the successful approach of the drug design. Due to the similarity of ligand interactions between MPO and serotonine transporter, the selectivity of this inhibitor was also tested on the serotonin transporter providing a selectivity index of 14 (KiSERT/IC₅₀MPO).

Full text of DOI:10.1016/j.ejmech.2016.07.053

European Journal of Medicinal Chemistry 123 (2016) 746e762
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel bis-arylalkylamines as myeloperoxidase inhibitors: Design,
synthesis, and structure-activity relationship study
a
a
a
e
c
ꢀ
Iyas Aldib , Michel Gelbcke , Jalal Soubhye , Martine Prevost , Paul G. Furtmüller ,
Christian Obinger c, Betina Elfving d, Ibaa Chikh Alard f, Goedele Roos e,
Cedric Delporte b, Gilles Berger a, Damien Dufour a b, Karim Zouaoui Boudjeltia g,
a,
,
ꢀ
a
,
, b, *
Jean Neve b, Francois Dufrasne a, Pierre Van Antwerpen a
ꢁ
a
ꢀ
ꢀ
Laboratoire de Chimie Pharmaceutique Organique, Faculte de Pharmacie, Universite Libre de Bruxelles, Brussels, Belgium
b
ꢀ
Analytical Platform of the Faculty of Pharmacy, Universite Libre de Bruxelles, Brussels, Belgium
c Department of Chemistry, Division of Biochemistry at the Vienna Institute of BioTechnology, BOKUdUniversity of Natural Resources and Life Sciences,
Muthgasse 18, A-1190, Vienna, Austria
d Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark
e
ꢀ
Laboratoire de Structure et Fonction des Membranes Biologiques, Universite Libre de Bruxelles, Brussels, Belgium
f
ꢀ
ꢀ
ꢀ
Laboratoire de Pharmacie Galenique et de Biopharmacie, Faculte de Pharmacie, Universite Libre de Bruxelles (ULB), Brussels, Belgium
Laboratory of Experimental Medicine, A. Vesale Hospital, Faculty of Medicine, Universite Libre de Bruxelles, Montigny-le-Tilleul, Belgium
g
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Human myeloperoxidase (MPO) plays an important role in innate immunity but also aggravates tissue
damage by oxidation of biomolecules at sites of inflammation. As a result from a recent high-throughput
virtual screening approach for MPO inhibitors, bis-2,20-[(dihydro-1,3(2H,4H) pyrimidinediyl)bis(-
methylene)]phenol was detected as a promising lead compound for inhibition of the MPO-typical two-
Received 13 May 2016
Received in revised form
5 July 2016
Accepted 22 July 2016
Available online 25 July 2016
electron oxidation of chloride to hypochlorous acid (IC50 ¼ 0.5
mM). In the present pharmacomodulation
study, 37 derivatives of this lead compound were designed and synthesized driven by comprehensive
docking studies and the impact on the chlorination activity of MPO. We describe the structural re-
quirements for optimum (i) binding to the heme periphery and (ii) inhibition capacity. Finally, the best
three inhibitors (bis-arylalkylamine derivatives) were probed for interaction with the MPO redox in-
termediates Compound I and Compound II. Determined apparent bimolecular rate constants together
with determination of reduction potential and nucleophilicity of the selected compounds allowed us to
propose a mechanism of inhibition. The best inhibitor was found to promote the accumulation of inactive
form of MPO-Compound II and has IC50 ¼ 54 nM, demonstrating the successful approach of the drug
design. Due to the similarity of ligand interactions between MPO and serotonine transporter, the
selectivity of this inhibitor was also tested on the serotonin transporter providing a selectivity index of 14
(KiSERT/IC50MPO).
Keywords:
Myeloperoxidase selective inhibitors
Bis-arylalkylamines derivatives
Reversible inhibitors
SERT
Pharmacomodulation
Docking
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
Myeloperoxidase (MPO, EC 1.11.2.2) is a heme enzyme belonging
to Family 1 of the peroxidase-cyclooxygenase superfamily [1]. It is
Abbreviations: MPO, myeloperoxidase; HDL, high density lipoprotein; LDL, low
density lipoprotein; NSAIDs, nonsteroidal anti-inflammatory drugs; SHA, sali-
cylhydroxamic acid; SERT, serotonin transporter; HTVS, high throughput virtual
screening; DMSO, dimethyl sulfoxide; TEA, trimethylamine; Eꢀ0, standard reduction
potential; E0, reduction potential; NHE, normal hydrogen electrode; RT, room
temperature; DCM, dichloromethane; EtOAc, ethyl acetate.
expressed mainly in human neutrophils and to some extent in
monocytes [2e6] and activates hydrogen peroxide to oxidize a
great variety of organic substrates to free radicals. However, what
sets MPO apart from other mammalian heme proteins is its
exceptional ability to oxidize chloride to hypochlorous acid, a
strong oxidant that kills bacteria and is toxic to human cells. So far
MPO is the only known heme protein with three heme to protein
* Corresponding author. Laboratoire de Chimie Pharmaceutique Organique, Fac-
ꢀ
ꢀ
ulte de Pharmacie, Universite Libre de Bruxelles, Brussels, Belgium.
E-mail address: pvantwer@ulb.ac.be (P. Van Antwerpen).
http://dx.doi.org/10.1016/j.ejmech.2016.07.053
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
747
covalent linkages (two ester bonds and one electron withdrawing
sulfonium ester linkage). These posttranslational modifications
distort the prosthetic group from planarity and lend an extraordi-
nary one- and two-electron oxidation capacity to the relevant
redox intermediate Compound I [7e9].
(3-phenoxypropyl)-1H-purine-2,6-dione (Fig. 1). The latter is a
xanthine structure which is related to the thioxanthine derivatives
developed by AstraZeneca [59], while the hexahydropyrimidine
structure was a novel chemical scaffold [32].
The purpose of the present study was to use the hexahy-
dropyrimidine structure as a basis for pharmacomodulation in or-
der to improve the potency in MPO inhibition. We first describe (i)
the strategy of design driven by comprehensive docking studies, (ii)
the chemical synthesis of these compounds as well as (iii) their
impact on the chlorination activity of MPO. Finally, we propose a
mechanism of inhibition based on these observations in combina-
tion with multi-mixing stopped-flow spectroscopic studies.
Neutrophils are innate immune cells that contain granules
loaded with antimicrobial peptides and proteins including MPO.
When neutrophils encounter pathogenic bacteria, they trap them
within intracellular vacuoles called phagosomes, into which they
discharge their granule contents. Simultaneously dioxygen is
reduced to superoxide that dismutates and delivers hydrogen
peroxide for the initiation of MPO activity and, finally, the pro-
duction of antimicrobial hypochlorous acid/hypochlorite (HOCl/
ClOꢁ) [10e14]. Myeloperoxidase may also kill bacteria outside of
neutrophils. Phagocytic and inflammatory stimuli induce neutro-
phils to cast out web-like structures consisting of DNA, histones and
granule proteins including MPO. These neutrophil extracellular
traps (NETS) also contribute to trapping and killing bacteria [15].
However, interest in MPO exploded when it was found to
aggravate tissue damage by oxidation of lipids, (lipo)proteins or
DNA at sites of inflammation [16e18]. It has been reported that
MPO plays a role in ischemia, and neurodegenerative diseases such
as multiple sclerosis and Parkinson disease [17,19e24], as well as in
sepsis [25], lung disease [26], atherosclerosis [27] and atrial fibril-
lation [28]. For example, in cardiovascular diseases MPO mediates
the production of proatherogenic lipoproteins (oxidized LDL and
HDL) promoting endothelial dysfunction, atheroma plaque forma-
tion, thrombosis, and ventricular remodeling [29,30]. The MPO/
H2O2/Clꢁ system seems to be involved in initiation and propagation
of inflammation during the development of atherosclerotic lesion
[31]. Indeed, Mox-LDL (LDL oxidized by MPO) has been shown to
initiate inflammatory responses in monocytes and endothelial cells
[32]. Myeloperoxidase takes also a part in transforming high-
density lipoprotein (HDL) by the selective modification of apoli-
poprotein A-1 to dysfunctional HDL [32e34].
2. Results
2.1. Docking experiments
Based on HTVS bis-2,20-[(dihydro-1,3(2H,4H)-pyrimidinediyl)
bis(methylene)]phenol (A1) (Fig. 1) was selected as a lead com-
pound as it was demonstrated to inhibit the chlorinating activity of
MPO (IC50 ¼ 0.5
mM) [32]. The X-ray structures of human MPO in
complex with cyanide and thiocyanate (PDB code 1DNW) as well as
in complex with HX1 (PDB code 4C1M) were used as target
structures in the docking studies.
The reliability of our docking procedure was previously assessed
by comparing the docking of SHA with its position in the crystal
structure of the MPO complex [61]. We validated further this pro-
cedure by docking 2-(3,5-bistrifluoromethylbenzylamino)-6-oxo-
1H-pyrimidine-5-carbohydroxamic acid (HX1) (Fig. 1) in the Xray
structure of the complex (PDB ID: 4C1M) [52,60] and as well as in
the other target Xray structure (PDB ID: 1DNW). The best pose of
HX1 in 4C1M, as determined by its lowest root-mean square de-
viation computed on all heavy atoms (RMSD) of 0.39 Å, is very
similar to its crystal position. (Fig. 2AeB). HX1 forms several non-
covalent interactions: a
p-stacking with pyrrole ring D and
Consequently, blocking the activity of MPO is a potential phar-
macological strategy for prevention and treatment of a broad range
of inflammatory diseases [35]. In the beginning upon random
chemical screening several natural compounds and commercial
drugs with antioxidant, anti-inflammatory and antihistaminic ac-
tivity were shown to inhibit MPO activity, including flufenamic acid
and indomethacin [36], nonsteroidal anti-inflammatory drugs
(NSAIDs) [37], e.g. diclofenac [38], flavonoids and polyphenols [39],
chalcones [40e42], resveratrol [43], caffeic acid, ferulic acid and
gallic acid [44]. Furthermore, indole and tryptamine derivatives
carrying an amine or an amide group on the carbon side chain
connected to the indole ring [45e47], salicylhydroxamic acid (SHA)
and hydrazide derivatives [48], isoniazide [49], dapsone [50], thi-
oxanthine derivatives [51], aromatic hydroxamates derivatives [52]
and indazolone analogs [53] were demonstrated to block MPO
activity.
Certainly, random screening was a very important step in the
process of discovering MPO inhibitors. But more recently, high
throughput virtual screening (HTVS) could be applied since high
resolution crystal structures of human MPO and MPO complexes
became available [54e57]. The first HTVS for MPO inhibitors based
on the interactions of SHA with active site residues was performed
in 2011 resulting in the selection of nine benzoic acid hydrazide
derivatives from Zinc database. The activity of those selected
compounds did not exceed that from SHA [58]. Finally, Aldib et al.
[32] uncovered eight novel and different (positively charged)
scaffolds of potential MPO inhibitors which were predicted to bind
close to Glu102 at the active site of MPO. The best inhibitors were:
A1, bis-2,20-[(dihydro-1,3(2H,4H)-pyrimidinediyl)bis(methylene)]
phenol, and F9, 8-[(2-aminoethyl)amino]-3,7-dihydro-3-methyl-7-
hydrogen bonds to Arg239, Gln91 and His95 with the ketone group
of its pyrimidin-(H)-one ring, its amide carbonyl and hydroxyl
group of the hydroxamic function, respectively. A stacking similar
to that observed for HX1 is also found in the crystal structure of
SHA-MPO complex [60] as well as in its docked position (Fig. 2D) in
addition to hydrogen bonds formed with Arg239, Gln91 and His95.
Similar stacking positions with pyrrole ring D were also observed in
docked positions of 5-fluorotryptamine and 3-(aminoalkyl)-5-
fluoroindole derivatives [45,46,61]. The poses of HX1 in 1DNW
(Fig. 2C) are less similar as indicated by a RMSD of 4.1 Å. However,
this higher RMSD value is mainly attributed to the bistri-
fluoromethylbenzyl moiety as shown by a RMSD of 0.7 Å calculated
by removing this group. The differences observed in both target
structures could be attributed to the better structural comple-
mentarity between the MPO conformation and HX1 in the 4C1M
structure. In any case, these docking data clearly underline that the
applied docking procedure provides a good basis for the planned
pharmacomodulation study.
Next we probed docking of A1 [32] (Fig. 2) to the two target
structures (Fig. 2EeF). In both cases, A1 shows almost the same
poses. In detail, the best-score position of A1 features a p-stacking
of one phenol ring (ring A) of A1 with the pyrrole ring D as well as
formation of a H-bond between the OH-group of this phenol ring
and Arg239. Furthermore, an ionic interaction between Glu102 and
the positively charged nitrogen of A1 is established in addition to
H-bonding between Glu102 and Phe147 with the OH-group of the
other phenol ring.
Based on the observed binding modes of A1 (Fig. 2), different
approaches were developed for the design of derivatives. In the
modulation process we focused mainly on the substitution of the

748
I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
Fig. 1. Structures of A1, HX1, SHA, S1, 5-fluorotryptamine, 3-(aminoalkyl)-5-fluoroindole derivatives and F9.
Fig. 2. Positions of HX1, SHA and A1 in MPO structures. (A) X-ray structure of human MPO in complex with HX1 (PDB ID: 4C1M). (B) Docked pose of HX1 in the X-ray MPO
structure (PDB ID: 4C1M). (C) Docked pose of HX1 in the X-ray MPO structure (PDB ID: 1DNW). (D) Docked pose of SHA in the Xray MPO structure (PDB ID: 4C1M). (E) Docking pose
of A1 in the X-ray MPO structure (PDB ID: 4C1M). (F) Docking pose of A1 in the X-ray MPO structure (PDB ID: 1DNW). Hydrogen bonds, cation-
as yellow, green and blue dotted lines. The ligands are shown as thick lines and the interacting residues as well as the heme as thinner lines. (For interpretation of the references to
p
and
p p interactions are depicted
ꢁ
colour in this figure legend, the reader is referred to the web version of this article.)

I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
749
Table 1
aromatic rings A and B. All designed compounds were docked in the
List of all synthesized compounds including IC50 values (
m
M) values and standard
two target structures. For the sake of simplicity, only the poses
obtained in 4C1M are described unless otherwise specified. In most
cases the docked poses are similar for both targets. Fig. 2.
reduction potentials, Eꢀ0 (mV). N.A., no activity; N.D., no data available. N.I.R., not in
range, i.e. >1.2 V.
2.1.1. The role of hydroxyl groups on aromatic rings A and B
Upon elimination of the hydroxyl groups on rings A and B of A1
(Fig. 1) compound 2 was designed and docked to both MPO re-
ceptor structures. Compared to A1 (ꢁ8.1 kcal/mol in 4C1M) the
calculated free energy upon binding of the best pose decreases
to ꢁ5.4 kcal/mol. In 4C1M, in addition to an aromatic stacking with
the pyrrole, the observed interactions between the ligand and the
0
Compound
R
X
IC50
Eꢀ0
A1
2
3
OH
H
H
CH2
CH2
C¼O
0.5 0.1
N.A
N.D
N.I.R
N.I.R
N.A
protein are a cation-p with Arg239, p-p interaction with His95 and
an ionic bond between Glu102 and the tertiary amine in ring C.
(Table 2 and Supplementary materials).
Introduction of a ketone function on the methylene group of
ring C in compound 2 aiming at increasing its stability toward hy-
drolysis [62], i.e. compound 3, results in a docked pose featuring a
Compound
R1
R2
R3
IC50
Eꢀ0
stacking with the pyrrole and one cation-p with Arg239 as in
4
5
6
7
8
OH
F
H
H
H
H
H
F
H
H
H
H
H
F
1.4 0.2
>10
>10
>10
>10
1072
1100
1062
1017
996
compound 2. No H-bonds are observed (Table 2 and Supplementary
materials).
2.1.2. Shifting the position of the amino groups in ring C and
introduction of fluorine
H
Changing the positions of amino groups in compound A1
through replacing hexahydropryrimidine ring by piperazine ring
should increase the hydrolytic stability [62,63] and leads to a
decrease in the predicted binding energy. Interactions formed by
compound 4 are roughly similar to those of A1 except for the hy-
droxyl group of the ring that is not stacked onto the pyrrole and
Compound
X
Y
IC50
9
10
N
CH
CH2
NH
>10
>10
953
960
which lacks hydrogen bonds (Table
materials).
2
and Supplementary
Further modification of compound 4 was achieved by substi-
tution of the OH-function in rings A and B by fluorine (compound 5)
or elimination of the OH-group and introduction of fluorine at
different positions (compounds 6 and 7) or elimination of both OH-
groups (compound 8) (Table 1). Fluorine is often used to substitute
OH-groups since it can also establish polar interactions and in
addition increases the metabolic stability of molecules [64]. Intro-
duction of fluorine substituents slightly decreases the predicted
affinity of the ligands. Generally, for compounds 5e8, Glu102 and
Compound
R1
R2
R3
R4
n
IC50
Eꢀ0
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
F
F
2
3
2
3
2
3
2
2
3
3
2
2
3
3
2
2
3
3
2
2
3
3
2
2
3
3
0.7 0.1
1.2 0.6
N.A.
973
928
N.I.R.
N.I.R.
948
902
907
870
871
844
N.I.R.
897
N.I.R.
825
N.I.R.
856
N.I.R.
833
N.I.R.
865
N.I.R.
869
N.I.R.
807
Arg239 with A1 establish an ionic interaction and a cation-
p
H
N.A.
interaction respectively but no hydrogen bonds are observed.
(Table 2 and Supplementary materials).
OH
OH
OH
OH
OH
OH
H
H
H
H
F
F
F
F
H
H
H
H
H
0.67 0.16
0.37 0.06
1.85 0.01
0.65 0.02
2.5 0.8
0.35 0.05
N.A.
2.70 0.01
N.A.
0.36 0.11
N.A.
0.70 0.06
N.A.
0.27 0.03
N.A.
1.16 0.05
N.A.
0.49 0.12
N.A.
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OH
OCH3
OH
2.1.3. Elimination of ring B and of an amino group in ring C
Compounds 9 and 10 lack both the aromatic ring B as well as one
of the tertiary amino groups of ring C to study the role of ring A and
one amino group (Table 1). Compound 9 (with smaller distance
between OH and amino group) and 10 both show a stacking though
shifted for compound 9 and exhibit a substantial decrease in the
predicted affinity relative to A1. They formed interactions similar to
A1 apart from those arising from the second hydroxylated ring (see
Table 2 and Supplementary materials).
F
F
F
H
H
H
H
2.1.4. Opening heterocycle C
This modulation was done in order to test the effect of removal
of the heterocycle C. However, the amine function was kept as it
was demonstrated to be beneficial for inhibition of MPO [45,47].
Next we probed the length of the bridge between this amino group
and aromatic ring B as well as the impact of substitutions on rings A
and B (Table 1). With increasing lengths of the bridge both the
lipophilicity and the flexibility of the ligand could be raised. In
compounds 11 and 12, the benzyl moiety makes a T-shaped inter-
action with or shifted stacking on pyrrole D while the hydroxyl
OCH3
OH
OCH3
OH
H
H
H
F
F
F
0.66 0.17
N.A.
0.37 0.05
N.I.R.
820
(continued on next page)

750
I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
Table 1 (continued )
stacking of one of their aromatic moiety on pyrrole D. A cation-
p
makes with Arg239 is also a rather recurrent interaction. Although
it is always perilous to rank the ligand affinity using the scoring
functions one can however notice that compound 38 is the com-
pound predicted to have the best score.
Compound
n
IC50
Eꢀ0
2.2. Chemistry
Schemes 1e4 summarize the procedures for syntheses of com-
pounds 1e38. Synthesis of the hexahydropyrimidine derivative 2
was based on a reductive amination reaction [65,66] followed by a
condensation reaction with formaldehyde. For synthesis of com-
pound 3 different reaction conditions [67] were chosen (Scheme 1).
For synthesis of piperazine [68] derivatives different aldehydes
(compounds 4e7) or benzylchloride [69] (compound 8) were used
(Scheme 2). The piperidine derivative 9 was synthesized by same
method used for piperazine derivatives, while other piperidine
derivative 10 was obtained by demethylation of 3-(2-
methoxybenzyl)piperidine [70,71](Scheme 3). The pathways fol-
lowed for the syntheses of bis(arylalkyl)amine derivatives (com-
pounds 11e36) and the indole derivatives (37, 38) are summarized
in Scheme 4. They include either a reductive amination step or two
steps consisting of a reductive amination followed by a demethy-
lation reaction [65,66,70,71] (Scheme 4).
Compound
n
IC50
Eꢀ0
37
38
2
3
0.085 0.017
0.054 0.007
902
872
group kept on aromatic ring A and the amine groups makes in-
teractions similar to those of A1 (Table 2 and Supplementary
materials). Upon elimination of the hydroxyl group (i.e. com-
pounds 13 and 14) these hydrogen bonds are lost and the affinity
scores are decreased by about 1.5 kcal molꢁ1 compared to com-
pounds 11 and 12 (Table 2). A cation-p interaction is however forms
with the aromatic moiety stacked on pyrrole D and Arg239.
Keeping one hydroxyl group on aromatic ring A and replacing
the other one on aromatic ring B by fluorine together with either an
ethyl or propyl bridge lead to compounds 15 and 16 (Table 1). Their
docking poses show stacking or shifted stacking poses of the fluo-
robenzyl group on pyrrole D and an ionic interaction with Glu102
(Table 2). Keeping the hydroxyl group on aromatic ring A and
having on ring B the OH-group or a methoxy group together with
either an ethyl or propyl bridge give compounds 17e20 (Table 2,
Fig. 3A). Compounds 17 and 19 (carrying the methoxy group)
feature a slightly more favorable free energy of binding relative to
the compounds carrying an eOH-group (Table 2).
Removing the hydroxyl group on aromatic ring A and keeping
either a hydroxyl or methoxy group on ring B together with either
an ethyl or propyl bridge lead to compounds 21e24 (Table 1). In
contrast to compounds 21 and 23 (carrying the methoxy group),
compounds 22 and 24 feature a better stacking onto pyrrole D and
have their eOH-groups hydrogen bonding to Arg239 (Table 2
Supplementary materials).
2.3. Inhibition of chlorination activity of myeloperoxidase
For assessment of inhibition of the chlorination activity of MPO
(i.e. the production of hypochlorous acid) the taurine chloramine
assay was used in a high-throughput screening mode. Table 1
presents the respective IC50 values for the 37 synthesized com-
pounds. The IC50 value for the lead compound A1 was 500 nM. The
data clearly underline the importance of the hydroxyl groups on the
aromatic rings A and B for inhibition of taurine chlorination. Except
compound 4 all piperazine derivatives without a hydroxyl group
(i.e. compounds 5e8) were poor inhibitors. This was also the case
for compounds 9 and 10 that lack one of the two aromatic rings.
By contrast the best inhibitors were those with 5-fluoroindole
instead of aromatic ring B with IC50 values of 85 nM (compound
37) and 54 nM (compound 38), respectively. The latter has the
longer (propyl) bridge between the amino function and the indole
ring. Further compounds with IC50 values lower than A1 include
propylamine compounds (16, 20, 24, 28, 32, and 36). Importantly,
their longer bridge (propyl) chain gives more flexibility between
the amino group and the aromatic ring B. They have also a fluorine
(compound 16) or hydroxyl group (compounds 20, 24, 28, 32, and
36) at position R4 on aromatic ring B. As demonstrated in Table 1
compounds that have a methoxy group at position R4 on the aro-
matic ring B and fluorine on aromatic ring A or compounds which
have no hydroxyl groups on two rings A, B were unable to block the
chlorination activity of MPO as with 13, 14, 21, 23, 25, 27, 29, 31, 33,
and 35. Furthermore the position of fluorine on aromatic ring A
seems to modulate the inhibition capacity of those compounds to
some extent with compound 28 (ortho position) being the most
effective of this series. Note that exchange of the hydroxyl group
(ortho position) on aromatic ring A (18, 20) by fluorine (26, 28)
decreases the respective IC50 values (Table 1). Some ethylamine
derivatives (compounds 11, 15, 17, 18, 22, 26, 30, and 34) and pro-
pylamine derivatives (only hydroxyl on ring A or one methoxy on
ring B and one hydroxyl on A as 12, 19) also showed reasonable
inhibition of MPO-mediated halogenation of taurine but their
respective IC50 values were higher than that of the A1.
Further modulation included (i) replacement of the hydroxyl
group on aromatic ring A by fluorine in ortho (R1), meta (R2) or para
(R3) positions, (ii) having either a hydroxyl or methoxy group (R4)
on aromatic ring B, and (iii) using ethyl or propyl as bridge (com-
pounds 25e36, Table 1). Docking experiments show that all com-
pounds carrying
a hydroxyl group on ring B feature their
fluorobenzyl moieties stacked onto the pyrrole D whereas in
compounds carrying the stacking onto the heme is provided either
by the a methoxy- or the fluorobenzyl group. Compared to A1 the
predicted affinities are lower for all compounds (Table 2).
The last two compounds 37 and 38 were designed by hybridi-
zation of the hit A1 and 5-fluorotryptamine where hydroxyl group
in aromatic ring A (from A1) is linked to 5-fluoroindole (from 5-
fluorotryptamine), using ethyl or propyl as linker (Table 1). Both
compounds exhibit an ionic interaction with Glu102 and hydrogen
bonds with Phe147 and Glu102 and a cation-
p with Arg239.
Remarkably the predicted affinity of compound 37 is as high as for
A1 (Table 2).
To sum up all designed compounds have in common to form an
ionic interaction between Glu102 and their amine group and a

I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
751
Table 2
List of all compounds along with the predicted affinity (
DGscore (kcal/mol)) of their best score docked positions and their interactions made with the receptor structure (PDB
code: 4C1M). HB, hydrogen bonds; SB, ionic interaction or salt bridge; Cat-
shifted stacking, TS, T-shaped interaction).
p, cation-
p;
pꢁ
p, interaction
pꢁp; Stacking, stacking with one heme pyrrole, (S, full stacking; SS,
Compound
Receptor 4C1M
Gscore
D
Compound-receptor interactions
HB
SB
Cat-
p
p
ꢁp
Stacking
S
HX1
ꢁ6.4
ꢁ8.1
Gln91
e
His95
Arg239
A1
Arg239
Glu102
Arg239
S
Glu102
Phe147
Heme propionate
2
3
4
ꢁ5.4
ꢁ4.1
ꢁ6.6
e
Glu102
e
Arg239
Arg239
Arg239
His95
S
S
S
e
Arg239
Heme propionate
Glu102
5
6
ꢁ5.2
ꢁ5.7
e
Glu102
Glu102
Arg239
Arg239
Arg424
Arg239
Arg239
Arg239
Arg239
SS
S
e
7
8
9
10
ꢁ4.8
ꢁ5.3
ꢁ4.4
ꢁ5.6
e
Glu102
Glu102
Glu102
Glu102
S
S
SS
S
e
Heme propionate
Arg239
Heme propionate
Glu102
Phe147
Glu102
11
12
ꢁ6.3
ꢁ6.9
Glu102
Glu102
SS
TS
Phe147
13
14
15
ꢁ4.9
ꢁ5.3
ꢁ6.0
e
Glu102
Glu102
Glu102
Arg239
Arg239
S
S
SS
e
Phe147
Glu102
16
17
ꢁ5.4
ꢁ7.6
Heme propionate
Glu102
Phe147
Glu102
Glu102
Arg239
His95
S
SS
18
19
ꢁ6.5
ꢁ7.2
Heme propionate
Glu102
Heme propionate
Glu102
Arg239
Arg239
Arg239
SS
S
Arg239
His95
Heme propionate
Heme propionate
e
20
21
ꢁ6.3
ꢁ5.8
Glu102
Glu102
Heme propionate
Glu102
SS
SS
Phe147
22
23
ꢁ6.0
ꢁ5.7
Arg239
Heme propionate
Arg239
Arg424
Arg239
S
e
Glu102
Heme propionate
Glu102
Glu102
Heme propionate
Glu102
Glu102
Heme propionate
Glu102
Heme propionate
Glu102
Glu102
SS
24
25
ꢁ5.9
ꢁ5.9
Arg239
e
Arg424
S
SS
26
27
ꢁ5.7
ꢁ5.8
Glu102 Phe147
e
Arg239
Arg239
SS
S
His95
28
ꢁ5.9
Pro145
Arg239
Arg239
S
29
30
ꢁ5.2
ꢁ5.9
e
SS
SS
Glu102
Phe147
Arg239
Pro145
31
32
ꢁ5.1
ꢁ5.8
Glu102
Glu102
Heme propionate
Glu102
Glu102
Glu102
Glu102
Heme propionate
Glu102
Arg239
Arg239
S
SS
33
34
35
36
ꢁ5.4
ꢁ5.9
ꢁ6.5
ꢁ5.9
Arg239
Heme propionate
Arg239
Arg239
Arg239
Arg239
SS
SS
S
Pro145
SS
37
38
ꢁ6.2
ꢁ8.3
Glu102 Phe147
Heme propionate
Glu102 Phe147
Arg239
Arg239
Arg239
S
S
Glu102
His95
Heme propionate

752
I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
Fig. 3. Docked poses of compounds 20, 28, 38 in the Xray MPO structures. (A) Pose of compound 20 in the Xray MPO structure (PDB ID: 4C1M). (B) Pose of compound 20 in the
Xray MPO structure (PDB ID: 1DNW). (C) Pose of compound 28 in the Xray MPO structure (PDB ID: 4C1M). (D) Pose of compound 28 in the Xray MPO structure (PDB ID: 1DNW). (E)
Pose of compound 38 in the Xray MPO structure (PDB ID: 4C1M). (F) Pose of compound 38 in the Xray MPO structure (PDB ID: 1DNW). Hydrogen bonds, cation-
interactions are depicted as yellow, green and blue dotted lines. The ligands are shown as thick lines and the interacting residues as well as the heme as thinner lines. (For
p
and
p p
ꢁ
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Scheme 1. Synthesis of N-N0-1,3-bisbenzyl-1,2,3,4,5,6 hexahydropyrimidine and 1,3-dibenzyl-3,4,5,6-Tetrahydro-2(1H)-Pyrimidinone.
2.4. Transient-state kinetics of the reaction between MPO
Compound I and Compound II with inhibitors
reduce Compound I in two one-electron reduction steps via Com-
pound II to ferric MPO. Compound II is unable to oxidize halides
(see Fig. 4).
Next, the best inhibitors of MPO-mediated taurine chlorination,
i.e. compounds 20, 28 and 38 (see their docked poses in Fig. 3) were
subjected to a transient-state kinetic study using multimixing
stopped-flow spectroscopy [72]. Fig. 4 schematically represents the
halogenation and the peroxidase cycle of MPO. For initiation of both
pathways ferric MPO has to be oxidized by hydrogen peroxide to
Compound I. In the halogenation cycle Compound I oxidizes halides
to the corresponding hypohalous acids thereby being directly
reduced to ferric MPO. In the peroxidase cycle one-electron donors
For elucidation of the mechanism of MPO inhibition it is
important to know the mode of interaction of these molecules with
MPO Compounds I and II. Thus we preformed these redox in-
termediates in a sequential stopped-flow mode and probed their
reactivity towards compounds 20, 28 and 38 (Fig. 5 -Figure 6). All
three molecules efficiently reduce Compound I directly to Com-
pound II as it is demonstrated in Fig. 5A for the direct conversion of
Compound I mediated by compound 38. The monophasic reaction
was very fast and the obtained kobs values were linearly dependent

I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
753
Scheme 2. Synthesis of piperazine derivatives.
Scheme 3. Synthesis of phenylmethylpiperidine derivatives.
on the electron donor concentration thus allowing the calculation
of the apparent bimolecular rate constant (k2) at pH 7.0 (i.e.
1.4 ꢂ 107 Mꢁ1 sꢁ1) (Fig. 5B and C). In comparison to compound 38
the corresponding k2 values of compounds 20 and 38 were reduced
by ~60% and ~90%, respectively (Table 3).
more compared to S1 a non-selective MPO/SERT inhibitor (for
structure of S1 see Fig. 1). Other good inhibitors were more selec-
tive on MPO versus SERT than compound 38 but with lower activity
(compounds 20 and 28) (Table 3).
Compound II is also able to oxidize compounds 20, 28 and 38,
but the corresponding apparent bimolecular rate constants (k3) are
at least three orders of magnitude slower ranging between from
1.4 ꢂ 103 Mꢁ1 sꢁ1 to 4.8 ꢂ 103 Mꢁ1 sꢁ1 (Fig. 5DeF). As a conse-
quence, these compounds mediate the accumulation of Compound
II which is reflected by high k2/k3 ratios (Table 3). Kinetics study
demonstrated that the entire best active synthesized inhibitors
behaved as good one-electron donors for Compound I while their
rates of reaction with Compound II shift the reaction to the
peroxidase cycle and decreased the production of HOCl.
2.6. Electron density maps
Next, we calculated electron density maps and the nucleophi-
licity of compounds 20, 28 and 38 in order to predict the site of
oxidation (Supplementary materials). Obtained data demonstrated
that the oxidation happens preferentially at the benzene ring B
(compounds 20 and 28) or the indole ring (compound 38). This is
also reflected by the fact that the spin density of the oxidized
compounds is also located at benzene ring B or the indole cycle
(Fig. 7).
2.5. Serotonin reuptake inhibition
2.7. Determination of redox potentials
Because compound 38 carries an indole ring and has a structure
related to serotonin, it was tested for inhibition of SERT (together
with compounds 20 and 28 for comparison) [73] (Fig. 6) (Table 3).
Compound 38 is a weak inhibitor of SERT with a calculated
Finally, we determined the one-electron reduction potential of
the putative inhibitors by cyclic voltammetry (reference electrode
Ag/AgCl, 3 M NaCl; working electrode glassy carbon) in phosphate
buffer, pH 7.4. Eꢀ0 values were obtained by converting redox po-
tentials from Ag/AgCl, 3 M NaCl to normal hydrogen electrode NHE
by adding þ198 mV. The corresponding Eꢀ0 for most of the com-
pounds varied between 807 mV and 1100 mV (Table 1). For com-
pounds, which were unable to inhibit the chlorination activity of
MPO (compounds 2, 3, 13, 14, 21, 23, 25, 27, 29, 31, 33, 35) the
standard reduction potential could not be determined because the
Ki ¼ 0.8
mM, compared to 11 mM and 14 mM for compounds 20 and
28, respectively. The specificity index Ki/IC50 (SERT versus MPO) was
then measured. Lower Ki/IC50 values demonstrate dual inhibition
activity against both SERT and MPO, while higher values prove that
the synthesized compounds are selective for MPO, Ki/IC50 ¼ 14.8 of
compound 38 shows selectivity for MPO versus SERT 3.5 times

754
I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
Scheme 4. Synthesis of Bis-Arylalkylamine and indole derivatives.
Eꢀ0 values were out of the measuring range (>1.2 V). The standard
reduction potentials of the relevant redox couples of MPO have
been published to be 1.35 V (Compound I/Compound II) and 0.97 V
(Compound II/ferric MPO), respectively [72,74e76]. This clearly
demonstrates that the one-electron oxidation by Compound I of all
inhibiting compounds is favorable, although there is no clear cor-
relation between IC50 and Eꢀ’ values.
(compounds 11e38).
Upon substitution of aromatic ring A at positions R1, R2 or R3 by
either hydrogen, fluorine or the hydroxyl group as well as of aro-
matic ring B at position R4 with either hydrogen, fluorine, hydroxyl
or methoxy groups we aimed at investigating the importance of
these functionalities for making non-covalent interactions within
the substrate access channel and the heme cavity of MPO. Gener-
ally, this work clearly underlined the importance of the presence of
hydroxyl groups on both aromatic rings for maintenance of the
inhibition capacity. Independent of the nature of bridge between
rings A and B (piperidine or piperazine or linear bridge), com-
pounds without any hydroxyl group on the aromatic rings were
extremely poor inhibitors (compounds 5e10) or completely lost the
capacity to interfere with MPO activity (compounds 2, 3, 13, 14, 21,
23, 25, 27, 29, 31, 33 and 35, respectively). This might be related to
decreased binding affinity (e.g. loss of interactions formed by hy-
droxylated rings) and/or an increase in the standard reduction
potential of the corresponding compounds. This work clearly
demonstrated that the inhibition capacity is related to the ability of
the compounds to donate electrons to MPO Compound I. The
3. Discussion
Upon virtual screening of large databases [32] bis-2,20-[(dihy-
dro-1,3(2H,4H)-pyrimidinediyl)bis(methylene)]phenol (A1) was
detected to act as potential inhibitor of the chlorination activity of
MPO. In this work we used A1 as a lead compound for pharmaco-
modulation in order to improve its properties and understand the
mechanism of interaction and inhibition. The general structure of
the novel series of compounds comprised two aromatic rings A and
B (with hydrogen bonding groups on each) which are bridged by at
least one amino group either within a heterocyclic structure (ring
C) (A1 and compounds 2e10) or as part of an open aliphatic bridge

I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
755
Fig. 4. Peroxidase and halogenation cycle of myeloperoxidase. Upon reaction of the
ferric protein with hydrogen peroxide Compound I [þꢃPorFe(IV) ¼ O or oxoiron(IV)
porphyrin radical cation] is formed (Reaction 1). In the peroxidase cycle one electron
donors (AH2) reduce Compound I to Compound II (Reaction 2) and Compound II to the
ferric enzyme (Reaction 3). The transition of Compound
I to Compound II
[PorFe(IV) ¼ O or oxoiron(IV)] is also mediated by H2O2 as one-electron donor. In the
halogenation cycle Compound I is directly reduced by halides (Xꢁ) thereby releasing
hypohalous acids (HOX) (Reaction 4).
aromatic ring B linked to longer bridge has been shown to be the
main oxidation site. Having a methoxy group instead of a hydroxyl
group on ring B significantly increased the reduction potential of
the corresponding compounds thereby losing the inhibition ca-
pacity despite the fact that those compounds exhibited the same
interaction pattern and still bound to the heme cavity. Generally,
the impact of substitution of ring B on MPO inhibition is more
pronounced than that of ring A. Upon removing of aromatic ring B
(compounds 9 and 10) the inhibition capacity was almost lost.
Furthermore, our study revealed that (i) the presence of one
amino group on the bridge (independent of being part of a cyclic
structure or an aliphatic chain) between rings A and B is sufficient
for the establishment of binding to Glu102 and (ii) that the pres-
ence of three methylene groups between the secondary amine and
the aromatic ring B improved the inhibition of chlorination and
thus decreased the IC50 values. These results show that the hy-
droxyl group on ring B is more important than on ring A. Indeed,
the hydroxyl group in aromatic ring B in compound 24 increases
the activity by nearly four times compared to compound 12 which
has hydroxyl group on aromatic ring A. This indicates that the
distance between the hydrogen bonding group of ring B and the
amino group is very important. Additionally, the docking poses of
active compounds 26, 28, 30, 32, 34 and 36, show ionic and
hydrogen bonding interactions between Glu102 and hydroxyl
group on ring B while the smaller volume of fluorine on ring A
allows the stacking pose of ring A.
This is also observed in our new compounds 37 and 38 which
carry a fluoroindole ring instead of the aromatic ring B obtained by
hybridization of hit A1 and the potent MPO inhibitors fluoro-
tryptamine derivatives [45]. Compound 38 is the best inhibitor of
this study, its IC50 is 54 nM, which is 10-times lower compared to
the lead compound A1, despite showing similar interactions with
Glu102 and Arg239 as well as similar score values and similar
stacking compared to both HX1 and A1. Moreover, upon elimina-
tion of this hydroxyl group in compound 38 (Table 3, compound S1)
the inhibition capacity is improved (IC50 ¼ 13 nM) [45]. This could
be related with the architecture and composition of the substrate
access channel of MPO which at its entrance to the heme cavity
forms a funnel shaped hydrophobic pocket. Thus elimination of the
hydroxyl group or exchange by fluorine increases the hydropho-
bicity and improve the inhibition activity towards MPO. However,
Fig. 5. Reaction of MPO Compounds I and II with compound 38. (A) Spectral
changes upon mixing 1.0
25 ꢀC. (B) Typical time trace and single-exponential fit (red) at 456 nm for the reaction
between 1 M Compound I and 8 M compound 38. (C) Pseudo-first-order rate con-
stants for Compound I reduction plotted against concentration of compound 38. (D)
Spectral changes of reduction of 2 M Compound II by 250 M compound 38 at pH 7.4
and 25 ꢀC. (E) Typical time trace and single-exponential fit (red) of the reaction be-
tween 1 M MPO Compound II and 250 M compound 38 followed at 456 nm. (F)
mM MPO Compound I with 2 mM compound 38 at pH 7.4 and
m
m
m
m
m
m
Pseudo-first-order rate constants for the reduction of MPO Compound II plotted
against concentration of compound 38. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)

756
I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
Fig. 6. Structures of the most potent synthesized MPO inhibitors derived from compound A1.
Table 3
Apparent bimolecular rate constants of MPO Compound I (k2) and Compound II reduction (k3) by compounds 20, 28 and 38. Additionally, values of 5-HT reuptake inhibition
and MPO inhibition (IC50 in
m
M) are included. ND ¼ no data available.
Compound
Compound I reduction (Mꢁ1Sꢁ1
)
Compound II reduction (Mꢁ1Sꢁ1
)
Ratio of k2/k3
MPO IC50
(
m
M)
SERT Ki (
m
M)
SERT Ki/MPO IC50
20
28
38
S1a
1.5 ꢂ 106
5.7 ꢂ 106
1.4 ꢂ 107
ND
4.8 ꢂ 103
1.4 ꢂ 103
3.5 ꢂ 103
ND
313
0.35 0.05
0.27 0.03
0.054 0.007
0.013 0.002
11
14
2
3
31.4
51.8
14.8
4.1
4071
4000
ND
0.8 0.2
0.05 0.02
a
S1 has the same structure as compound 38 but lacks the hydroxyl group.
the introduction of the hydroxyl group is accompanied by a dra-
matic decrease of the SERT inhibition leading to an increase of the
selectivity of this compound as the selectivity increase from 4
(compound S1) to 14 (compound 38) (Table 3). It is also noteworthy
that the low IC50 at submicromolar range obtained in this study is
compatible with an enzyme inhibition and not a simple scavenging
effect towards hypochlorous acid. Indeed, the amount of MPO,
chloride and hydrogen peroxide in the MPO inhibition assay allow
E’ꢀ values less than 1.2 V which allows an oxidation of the molecule
by Compound I. As a consequence, the determined apparent
bimolecular rate constants for Compound I reduction (k3) of com-
pounds 20, 28 and 38 are shown to be about 3 magnitudes of order
higher compared to those for Compound II reduction k4). The
resulting high ratios of k3/k4 lead to accumulation of Compound II
which is the inactive form of MPO shifting the MPO from the
chlorination cycle to the peroxidase cycle (Fig. 4). Subsequently, our
inhibitors are reversible MPO inhibitors [32,35,78] and substrates
for MPO and must be quickly oxidized by Compound I of MPO to
accumulate Compound II. As mentioned above substitution of hy-
droxyl groups by methoxy groups and fluorine renders the corre-
sponding molecules to weak electron donors and thus poor MPO
inhibitors. All inactive compounds had reduction potentials >1.2 V.
Thus, the key structural elements of these good reversible MPO
inhibitors include: 1) Two aromatic rings with at least one hydroxyl
group, which is important for building hydrogen bonds with sur-
rounding residues. In this context, ring A and ring B are essential to
keep the activity but the hydroxyl group on ring A is less crucial
than the hydroxyl group on ring B since aromatic ring B linked to
longer bridge chain (3 carbons) allows oxidation site of those
molecules. 2) One amino group on the bridge between the two
rings for interaction with Glu102 that is crucial for optimum ligand
binding [3], and 3) An overall reduction potential
(0.85 V < Eꢀ < 1.2 V) that allows rapid conversion of Compound I to
Compound II in order to outcompete the two-electron reduction of
Compound I by chloride [3].
us to produce around 60 mM of hypochlorous acid while the in-
hibitors act at concentration 1000 fold less.
The present study also supports the difficulty to predict the
inhibitory effect on MPO using the scoring functions, regardless of
the receptor structure used, which can reasonably not discriminate
between the different ligands. One of the possible reasons is that
factors other than binding contribute in the MPO inhibition.
Docking calculations provide valuable information about binding
poses and non-covalent interactions but in case of this oxidore-
ductase also the redox chemistry of both the enzyme and the li-
gands are very important for inhibition. Our mechanistic study
clearly underlines that the inhibiting compounds act as one-
electron donors for Compound I and Compound II of MPO. This
was demonstrated for the best inhibitors, i.e. compounds 20, 28 and
38. Compound I of MPO is known to be one of the strongest
enzymatic oxidants with a standard reduction potential of 1.35 V
for the redox couple Compound I/Compound II. This compares with
0.97 V for the couple Compound II/ferric MPO, respectively
[72,75e77]. The results show that all of the active compounds have

I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
757
Fig. 7. Electron density calculations: HOMO of the ligands shows that the oxidation probably happens at the green spots of ring B or indole cycle. (A) Compound 20, (B)
Compound 28 (C) Compound 38. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Conclusion
toward MPO versus SERT compared to S1. Other compounds
namely compound 28 proved to be the most selective one toward
peroxidase (13 times more than compound S1) and 2 times more
active than the hit A1. Above all, the pharmacomodulation has
given new bis-arylpropylamine derivatives which were first intro-
duced as novel MPO inhibitors. These bis-arylpropylamine de-
rivatives are promising novel MPO inhibitors and it will be
interesting to probe their efficacy in in vivo model systems in the
future.
To sum up, new chemical entities for MPO inhibition were
found. Structure-based drug design has been successful to create
better inhibitors. They act as electron donors of the oxidoreductase
and efficiently shift MPO from the chlorination cycle to the
peroxidase cycle (Fig. 4) and thus act as reversible MPO inhibitors
[32,35,78]. Eight compounds were more active than the starting hit
A1 with submicromolar activities. The best compound 38 is 10
times more active than the starting hit with higher selectivity

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I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
5. Experimental section: materials and methods
5.1.1.3. Synthesis procedure of compound 3. To a solution of N,N0-
trimethyleneurea (0.50 g, 5 mmol) in 1,4-dioxane (5 ml) was added
to NaH 95% (0.24 g, 10 mmol) and benzyl chloride (1.27 g, 10 mmol)
dissolved in 45 ml of 1,4-dioxane at 0 ꢀC. The reaction mixture was
refluxed overnight and cooled to room temperature. The solvent
was evaporated under reduced pressure. CH2Cl2 was added, the
organic layer was washed with water, dried over anhydrous
Na2SO4, filtered and evaporated. The crude material was purified by
flash chromatography on silica gel SiO2 by using a mixture of
CH2Cl2/MeOH (95:5) to afford white solid (1.07g, yield 76%).
5.1. Synthesis
1H and 13C NMR spectra were taken on a Bruker Avance
300 M Hz spectrometer (Wissemburg, France) at 293 K. Chemical
shifts (d) are given in parts per million (ppm) relative to tetrame-
thylsilane (TMS), and the coupling constants are expressed in hertz.
Infrared spectroscopic analysis was performed with a Shimadzu
(Kyoto, Japan) IRAffinity-1 spectrophotometer equipped with ATR
system and the peak data are given in cmꢁ1. Mass spectrometric
data were obtained on a QTOF 6520 (Agilent, Palo Alto, CA), positive
mode, electrospray ionization (ESI), mode time of flight (TOF), by
diffusion of 0.5 mL/min, by mobile phase 0.1 M HCOOH/CH3OH
(50:50) (VCAP 3500 V, source T, 350 ꢀC; fragmentation, 110 V;
skimmer, 65 V). All reactions were followed by thin-layer chro-
matography (TLC) carried out on Fluka (Bornem, Belgium) PET foils
silica gel 60, and compounds were visualized by UV and by spraying
Van Urk reagent (0.125 g of p-dimethylaminobenzaldehyde dis-
solved in 100 mL of 65% sulfuric acid with addition of 0.1 mL of 5%
ferric chloride). Flash chromatography was performed with silica
5.1.1.4. General procedure of synthesis for compounds 4e7. A solu-
tion of anhydrous piperazine (0.86 g, 10 mmol) in EtOH (10 ml) was
added dropwise to solution of acetic acid (10 mmol) and the suit-
able aldehyde (20 mmol) in EtOH (40 ml), then sodium cyanobor-
ohydride NaBH3CN (1.26 g, 20 mmol) was added after 1 h, and the
resulting solution was stirred overnight. The solution was evapo-
rated and the residue was taken up with 20 ml of water and
extracted two times with 25 ml of CH2Cl2, the organic layer was
washed with water, dried over anhydrous Na2SO4, filtered and
evaporated. The crude material was purified by flash chromatog-
raphy using CH2Cl2/MeOH to afford the desired compound (see
Supplementary materials).
(63e200
mm) from MP Biomedicals (Santa Ana, CA) or with neutral
alumina (Al2O3, 50e200
mm) purchased from Merck. Organic so-
lutions were dried over Na2SO4 and concentrated with a Buchi
rotatory evaporator (Flawil, Switzerland). Starting materials: 3-(2-
methoxybenzyl)-piperidine was purchased from (Bio Block; USA(,
3-(2-fluorophenyl)-propylamine, 3-(2-ethylamine)-5-fluoro-1H-
Indole, 3-(3-propylamine)-5-fluoro-1H-Indole were purchased
from Enamine Building Blocks (Monmouth, Jct, USA), 3-(2-
methoxyphenyl)-propylamine was purchased from ChemBridge
Building Block Library (San Diego, CA, USA), piperazine was pur-
chased from TCI (Japan), 2-(2-methoxyphenyl)-ethylamine, 2-(2-
5.1.1.5. Synthesis procedure of compound 8. A solution of piperazine
(0.86 g, 10 mmol) in CH2Cl2 (10 ml) was added dropwise to solution
of triethylamine (2.22 g, 22 mmol) and benzylchloride (2.53 g,
20 mmol) in 40 ml of CH2Cl2, and the resulting suspension was
stirred overnight. The mixture was evaporated under reduced
pressure, the residue extracted by ethyl acetate EtOAc (50 ml), the
organic layer was washed with water, dried over anhydrous
Na2SO4, filtered and evaporated. The crude material was purified by
flash chromatography (silica gel SiO2) using EtOAc/MeOH (80:20).
The silicagel was taken up from the column, mixed with a mixture
of EtOAc/MeOH (60:40), filtered and the resulted solvent was
evaporated to afford white solid (0.47 g, yield 18%).
fluorophenyl)-ethylamine,
3-phenylpropylamine,
2-
phenylethylamine were purchased from (Sigma Aldrich, Bornem,
Belgium). Purity was determined with liquid chromatography (LC)
with diode-array detection (DAD) (Waters, Milford, MA, USA) on a
150 mm ꢂ 4.6 mm Symmetry C8 column at a mobile phase flow rate
of 1 mL/min. The mobile phase was a mixture of methanol (700 mL)
and a KH2PO4 solution (0.07 M in methanol 5%, 300 mL) adjusted to
pH 3.0 with a 34 wt % H3PO4 solution. The purity was ꢄ95% for all
compounds.
5.1.1.6. Synthesis procedure of compound 9. A solution of piperidine
(0.85 g, 10 mmol) and glacial acetic acid (0.60 g, 10 mmol) in EtOH
(40 ml) was added dropwise to a solution of salicylaldehyde (1.22 g,
10 mmol) in EtOH (10 ml). After 20 min NaBH3CN (1.26 g, 20 mmol)
was added and the reaction was left overnight. The reaction
mixture was acidified by a solution of HCl 1 N to arrive to pH ¼ 2.
The solvent was evaporated under reduced pressure, and 40 ml of
water was added and then a solution of KOH 4 N was added until
pH ¼ 7. The water solution was extracted with Et2O twice (30 ml)
and the organic solvent was washed by brine twice (20 ml), dried
over anhydrous Na2SO4, filtered and evaporated. The crude mate-
rial was purified by flash chromatography on silica gel SiO2
(gradient 90:10 to 80:20 EtOAc:NH3 in MeOH 10%) to afford white
pale solid (0.97 g, yield 51%).
5.1.1. Chemical synthesis procedures
5.1.1.1. Synthesis procedure of compound 1. To a solution of benz-
aldehyde (5 mmol, 0.51 g) in 50 ml of ethanol an ethanolic solution
of 1,3-diaminopropane (2.5 mmol, 0.19 g) was added dropwise
during 10 min at room temperature. After 2 h sodium cyanobor-
ohydride (10 mmol, 0.63 g) was added and left overnight. The so-
lution was evaporated and the residue was taken up with 10 ml of
water and extracted two times with 25 ml of CH2Cl2. The organic
layer was dried over anhydrous Na2SO4, filtered and evaporated.
The crude material was purified by flash chromatography on silica
gel by using a mixture of CH2Cl2/MeOH (95:5) to afford colorless oil
(0.61 g, yield 96%).
5.1.1.7. Synthesis procedure of compound 10. A solution of boron
tribromide in CH2Cl2 (1.03 g, 4.07 mmol) was added dropwise to a
solution of 3-(2-methoxybenzyl)-piperidine (0.31 g, 1.5 mmol) in
CH2Cl2 (50 ml) at ꢁ78 ꢀC. Then the temperature of the mixture let
to come back to RT over a period of 1h and the reaction was stirred
overnight. An aqueous saturated solution of NaHCO3 was added
dropwise at 0 ꢀC and then pH was adjusted to reach the range of
7e8 with the same solution, the organic layer was separated and
the water layer was extracted by CH2Cl2 (3 ꢂ 40 ml). The combined
organic layers were dried using (Na2SO4) and evaporated under
reduced pressure. Purification for the crude material was done by
flash chromatography on Al2O3 by using CH2Cl2/NH3 solution in
MeOH 1%/(95:5) to afford white solid (0.06 g, yield 20%).
5.1.1.2. Synthesis procedure of compound 2. To compound 1 (0.61,
2.25 mmol) dissolved in ethanol (50 ml) a solution of formaldehyde
37 wt % in H2O (0.23 ml, 4.5 mmol) was added dropwise at room
temperature and the reaction left overnight. The crude mixture was
evaporated, CH2Cl2 was added (25 ml) and the organic layer was
washed with water, dried over anhydrous Na2SO4, filtered and
evaporated. The crude material was purified by flash chromatog-
raphy on silica gel SiO2 by using a mixture of CH2Cl2/MeOH (99:1)
to afford white pale solid (0.46 g, yield 73%).

I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
759
5.1.1.8. General procedure of synthesis for compounds (11e16) (17,19,
21,23,25,27,29,31,33,35,37,38). A solution of an arylalkylamine or an
indole derivative (10 mmol) in EtOH (5 ml) was added dropwise to
a solution of aldehyde (10 mmol) in 50 ml of EtOH, after one hour
NaBH3CN was added (20 mmol) the resulting solution was stirred
overnight. The solution was evaporated and the residue was taken
up with water and extracted with CH2Cl2. The organic layer was
dried over anhydrous Na2SO4, filtered and evaporated. The crude
material was purified by flash chromatography by using silica gel
SiO2 (for defined eluent percentage see Supplementary materials)
to afford desired compound.
IC50 values were then determined by standard procedures, taking
into account the absence of hydrogen peroxide as 100% inhibition
and the absence of inhibitors as 0% inhibition.
5.4. Transient-state kinetics
Highly purified MPO with purity index (A430/A280) of at least
0.86 was purchased from Planta Natural Products (http://www.
planta.at/). Its concentration was calculated by use of
ε430 ¼ 91 mMꢁ1 cmꢁ1. Enzyme concentration was determined by
using the extinction coefficient 112,000 Mꢁ1 cmꢁ1 at 412 nm [83].
Hydrogen peroxide obtained from a 30% solution was diluted and
the concentration was determined by absorbance measurement at
240 nm, where the extinction coefficient is 39.4 Mꢁ1 cmꢁ1. Inhib-
itor stock solutions were prepared in dimethyl sulfoxide and stored
in dark flasks. Dilution was performed with 200 mM phosphate
buffer, pH 7.4, to a final DMSO concentration of 2% (v/v) in all assays
[84].
5.1.1.9. General procedure of synthesis for compounds (18, 20, 22, 24,
26, 28, 30, 32, 34, 36). A solution of boron tribromide in CH2Cl2
(4.4 mmol) was added dropwise to a solution of compound
resulting from the previous general procedure (bis-arylalkylamine
derivative) (1.5 mmol) in CH2Cl2 (50 ml) at ꢁ78 ꢀC and the tem-
perature of the mixture was left to arrive RT over a period of 1 h and
the reaction was stirred overnight. An aqueous saturated solution
of NaHCO3 was added dropwise at 0 ꢀC and then pH was adjusted to
reach a range of 7e8 with the same solution, then the reaction
mixture was extracted by CH2Cl2; the combined organic layers
were separated, dried over (Na2SO4), and evaporated under
reduced pressure. Purification for the crude material was done by
flash chromatography of silica gel (see Supplementary materials) to
afford desired compound.
The multimixing stopped-flow measurements were performed
with the Applied Photophysics (UK) instrument SX-18MV. When
100 mL was shot into a flow cell having a 1 cm light path, the fastest
time for mixing two solutions and recording the first data point was
1.3 ms. Kinetics were followed both at single wavelength and by use
of a diode-array detector. At least three determinations (2000 data
points) of pseudo-first-order rate constants (kobs) were performed
for each substrate concentration (pH 7.4, 25 ꢀC) and the mean value
was used in the calculation of the apparent second-order rate
constants, which were calculated from the slope of the line defined
by a plot of kobs versus substrate concentration. To allow calculation
of pseudo-first-order rates, the concentrations of substrates were at
least 5 times in excess of the enzyme.
5.2. Docking experiments
The X-ray structure of human MPO complexed to cyanide and
thiocyanate (PDB code 1DNW) and the human MPO complexed to
HX1 (PDB code 4C1M) were used as the receptors targets in
docking studies [52,56]. The X-ray water and other ligand mole-
cules were removed from the active site. The ligand input files were
prepared according to the following procedure. The initial 3D
structures of the ligands were generated with the Ligprep module
from Schrodinger [79] [32] and the ligand partial charges were
ascribed by use of the OPLS force field [80]. The Epik program was
used to predict different protonation states of all ligands [81].
Docking was performed with the program (version 6.1), which
approximates a systematic search of positions, orientations, and
conformations of the ligand in the receptor binding site by use of a
series of hierarchical filters (www.schrodinger.com). The Glide XP
docking protocol and scoring function were used. The remaining
parameters were set to their default values.
Conditions of MPO Compound I formation were described
recently [85]. Typically, 8 mM MPO was premixed with 80 mM H2O2,
and after a delay time of 20 ms, Compound I was allowed to react
with varying concentrations of alkylindole derivative in 200 mM
phosphate buffer, pH 7.4. The reactions were followed at the Soret
maximum of Compound II (456 nm). Compound II formation and
reduction could be followed in one measurement. The resulting
biphasic curves at 456 nm showed the initial formation of Com-
pound II (increase in absorbance at 456 nm) and its subsequent
reduction to native MPO by the alkylindole derivatives (decrease in
absorbance at 456 nm).
5.5. Assessment of 5HT reuptake inhibition
Inhibition assessment of 5-HT reuptake was performed as fol-
lows: The mammalian expression plasmid pcDNA3.1 containing
human 5-HT transporter (hSERT) cDNA has been described previ-
ously by Kristensen et al. [86]. HEK-293 MSR cells (Invitrogen) were
cultured as monolayers in Dulbecco's modified Eagle's medium
(BioWhitaker) supplemented with 10% fetal calf serum (Invi-
5.3. Taurine chlorination assay
The assay is based on the production of taurine chloramine
produced by the MPO/H2O2/Clꢁ system in the presence of a selected
inhibitor at different concentrations as described by Van Ant-
werpen et al., 2007 [82]. The reaction mixture contained the
trogen), 100 units/mL penicillin, 100
mg/mL streptomycin (Bio-
following reagents in a final volume of 200
buffer (pH 7.4, 300 mM NaCl), 15 mM taurine, compound to be
tested (up to 20 M), and a fixed amount of recombinant MPO
(6.6 L of MPO batch solution diluted 2.5 times, 40 nM). When
necessary, the volume was adjusted with water. This mixture was
incubated at 37 ꢀC and the reaction was initiated with 10.0
L of
H2O2 (100 M). After 5 min, the reaction was stopped by the
addition of 10 L of catalase (8 units/ L). To determine the amount
of taurine chloramine produced, 50 L of 1.35 mM solution of thi-
onitrobenzoic acid was added and the volume was adjusted to
300 L with water. Then the absorbance of the solutions was
m
L: 10 mM phosphate
Whitaker), and 6 g/mL Geneticin (Invitrogen) at 95% humidity, 5%
m
p(CO2), and 37 ꢀC. Cells were detached from the culture flasks by
Versene (Invitrogen) and trypsin/EDTA (BioWhitaker) treatment
for subculturing or seeding into white TC-microtiter plates (Nunc).
Transfection and measurement of [3H]5-HT (PerkinElmer Life Sci-
ences) uptake was performed as described by Larsen et al. [87],
except that HEK-293 MSR cells (Invitrogen) were used instead of
COS-1 cells.
m
m
m
m
m
m
m
5.6. Determination of redox potentials
m
measured at 412 nm with a microplate reader, and the curve of
absorbance as a function of inhibitor concentration was plotted.
The experiments were achieved in a conventional three-
electrode cell at 25
2
ꢀC, associated with a potentionstat

760
I. Aldib et al. / European Journal of Medicinal Chemistry 123 (2016) 746e762
peroxidase-cyclooxygenase superfamily: reconstructed evolution of critical
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Epsilon (BASi, West Lafayette, IN). The working electrode was a
glassy carbon disk polished with 0.05 mm alumina (Metkron)
before each run, for linear cyclic voltammetry measurements. The
auxiliary electrode was a platinum wire. The reference electrode
was Ag/AgCl (3 M NaCl). The inhibitors were dissolved in 0.1 M
phosphate buffer, pH 7.4. The solutions were diluted by the same
buffer until inhibitor concentration of 1 ꢂ 10ꢁ4 M. Cyclic voltam-
mograms were obtained by a single cycle performed at a scan rate
of 100 mV sꢁ1. For the scan rate studies, the scan rate was varied
from ꢁ200 to 1000 mV sꢁ1. Eꢀ0 values were obtained by converting
redox potentials from Ag/AgCl, 3 M NaCl to normal hydrogen
electrode (NHE) by adding þ198 mV. Because the oxidation of our
compounds was irreversible, it is difficult to determine their Eꢀ0
values. However, in our case Eꢀ0 values are slightly less than E0 [45].
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Calculations of electron density maps of three best selected
active compounds (20, 28, 38) have been conducted in order to
predict their oxidation sites, the non-condensed Fukui function
[88] f ꢁðrÞ, calculated [89] as the difference between the electron
densities of the ligand having N0 and N0-1 electrons
(f ꢁðrÞ ¼
r
ðrÞðN0Þ ꢁ ðrÞðN0 ꢁ 1Þ), indicates the site in a molecule
r
having the maximum nucleophilic power, and thus it indicates the
place from where the electron will preferentially be donated during
oxidation. f ꢁðrÞ is related to the highest occupied orbital (HOMO)
electron density of the molecule. In accordance with the location of
the HOMO orbital, the density difference plots giving f ꢁðrÞobtained
on selected compounds shows where the oxidation happens pref-
erentially (see Supplementary materials). The spin density, ob-
tained as the difference between the electron density of the alfa and
beta electrons were calculated on the radical form of the
compounds.
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ischemia reperfusion, Am. J. Pathol. 171 (2007) 1743e1752.
The geometries of selected (20, 28, 38) synthesized compounds
are optimized at the B3LYP/6-311G(d,p) level. Frequency calcula-
tions were performed to ensure a local minimum. The electron
densities and molecular orbitals are calculated at the B3LYP/6-
311G(d,p) level on the optimized geometries. All calculations are
performed with Gaussian09 [90].
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Author contributions
[19] D.K. Choi, S. Pennathur, C. Perier, K. Tieu, P. Teismann, D.C. Wu, V. Jackson-
Lewis, M. Vila, J.P. Vonsattel, J.W. Heinecke, S. Przedborski, Ablation of the
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disease in mice, J. Neurosci. Off. J. Soc. Neurosci. 25 (2005) 6594e6600.
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W.F. Reynolds, Aberrant expression of myeloperoxidase in astrocytes pro-
motes phospholipid oxidation and memory deficits in a mouse model of
Alzheimer disease, J. Biol. Chem. 284 (2009) 3158e3169.
The manuscript was written in a collaborative mode by all au-
thors. All authors have given approval to the final version of the
manuscript.
Acknowledgment
[21] W.F. Reynolds, M. Hiltunen, M. Pirskanen, A. Mannermaa, S. Helisalmi,
M. Lehtovirta, I. Alafuzoff, H. Soininen, MPO and APOEepsilon4 poly-
morphisms interact to increase risk for AD in Finnish males, Neurology 55
(2000) 1284e1290.
[22] W.F. Reynolds, J. Rhees, D. Maciejewski, T. Paladino, H. Sieburg, R.A. Maki,
E. Masliah, Myeloperoxidase polymorphism is associated with gender specific
risk for Alzheimer's disease, Exp. Neurol. 155 (1999) 31e41.
[23] Y. Sawayama, Y. Miyazaki, K. Ando, K. Horio, C. Tsutsumi, D. Imanishi,
H. Tsushima, Y. Imaizumi, T. Hata, T. Fukushima, S. Yoshida, Y. Onimaru,
M. Iwanaga, J. Taguchi, K. Kuriyama, M. Tomonaga, Expression of myeloper-
oxidase enhances the chemosensitivity of leukemia cells through the gener-
ation of reactive oxygen species and the nitration of protein, Leukemia 22
(2008) 956e964.
We thank the “Wallonie Bruxelles International” Agency (www.
wbi.be) (grant no.: SOR/2011/33449), Fonds David & Alice Van
ꢀ
Buuren and the FRS-FNRS for their support. Martine Prevost is a
“Maı^tre de Recherche“ at the FNRS (Belgian Scientific Research
ꢀ
Fund). Cedric Delporte and Jalal Soubhye are research fellows at the
FNRS. Additionally, the work was supported by the Austrian Science
Foundation (project P25538). Finally, we thank prof. Jean-Michel
Kauffmann for his support in the redox potential measurement.
[24] R.M. Nagra, B. Becher, W.W. Tourtellotte, J.P. Antel, D. Gold, T. Paladino,
R.A. Smith, J.R. Nelson, W.F. Reynolds, Immunohistochemical and genetic
evidence of myeloperoxidase involvement in multiple sclerosis,
J. Neuroimmunol. 78 (1997) 97e107.
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Myeloperoxidase in Organ Dysfunction and Sepsis, Intensive care medicine,
Springer Berlin Heidelberg, 2007, pp. 173e187.
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C.C. Winterbourn, 3-Chlorotyrosine as a marker of protein damage by mye-
loperoxidase in tracheal aspirates from preterm infants: association with
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.07.053.
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