Structure-based design, synthesis, and pharmacological evaluation of 3-(Aminoalkyl)-5-fluoroindoles as myeloperoxidase inhibitors

Article abstract of DOI:10.1021/jm1009988

Oxidized low-density lipoproteins (LDLs) accumulate in the vascular wall and promote local inflammation, which contributes to the progression of the atheromatous plaque. The key role of myeloperoxidase (MPO) in this process is related to its ability to modify APO B-100 in the intima and at the surface of endothelial cells. A series of 3-(aminoalkyl)-5-fluoroindole analogues was designed and synthesized by exploiting the structure-based docking of 5-fluorotryptamine, a known MPO inhibitor. In vitro assays were used to study the effects of these compounds on the inhibition of MPO-mediated taurine chlorination and oxidation of LDLs. The kinetics of the interaction between the MPO redox intermediates, Compounds I and II, and these inhibitors was also investigated. The most potent molecules possessed a 4- or 5-carbon aminoalkyl side chain and no substituent on the amino group. The mode of binding of these analogues and the mechanism of inhibition is discussed with respect to the structure of MPO and its halogenation and peroxidase cycles.

Full text of DOI:10.1021/jm1009988

J. Med. Chem. 2010, 53, 8747–8759 8747
DOI: 10.1021/jm1009988
Structure-Based Design, Synthesis, and Pharmacological Evaluation of 3-(Aminoalkyl)-5-fluoroindoles
†
as Myeloperoxidase Inhibitors
‡
§
‡,
^
^
Jalal Soubhye, Martine Pr ꢀe vost, Pierre Van Antwerpen, Karim Zouaoui Boudjeltia, Alexandre Rousseau,
#
#
^
^
‡
‡
Paul G. Furtm u€ ller, Christian Obinger, Michel Vanhaeverbeek, Jean Ducobu, Jean N ꢁe ve, Michel Gelbcke, and
Fran c- ois Dufrasne*
,
‡
‡
Laboratoire de Chimie Pharmaceutique Organique, Facult eꢀ de Pharmacie, Universit eꢀ Libre de Bruxelles, Brussels, Belgium,
Laboratoire de Structure et Fonction des Membranes Biologiques, Universit eꢀ Libre de Bruxelles, Brussels, Belgium,
Analytical Platform of the Faculty of Pharmacy, Universit eꢀ Libre de Bruxelles, Brussels, Belgium, Laboratory of Experimental Medicine,
§
^
#
CHU Charleroi, A. V eꢀ sale Hospital, Universit eꢀ Libre de Bruxelles, Montigny-le-Tilleul, Belgium, and Department of Chemistry,
Division of Biochemistry, BOKU;University of Natural Resources and Life Sciences, Vienna, Austria
Received June 15, 2010
Oxidized low-density lipoproteins (LDLs) accumulate in the vascular wall and promote local inflam-
mation, which contributes to the progression of the atheromatous plaque. The key role of myeloper-
oxidase (MPO) in this process is related to its ability to modify APO B-100 in the intima and at the
surface of endothelial cells. A series of 3-(aminoalkyl)-5-fluoroindole analogues was designed and
synthesized by exploiting the structure-based docking of 5-fluorotryptamine, a known MPO inhibitor.
In vitro assays were used to study the effects of these compounds on the inhibition of MPO-mediated
taurine chlorination and oxidation of LDLs. The kinetics of the interaction between the MPO redox
intermediates, Compounds I and II, and these inhibitors was also investigated. The most potent
molecules possessed a 4- or 5-carbon aminoalkyl side chain and no substituent on the amino group. The
mode of binding of these analogues and the mechanism of inhibition is discussed with respect to the
structure of MPO and its halogenation and peroxidase cycles.
4-10
Introduction
inflammatory conditions.
Several of these pathologies
a
present major problems for public health at a global level.
To investigate the involvement of MPO in cardiovascular
disease, we focused our attention on atherosclerosis. It is well-
known that MPO oxidizes the apolipoprotein (APO) B-100 of
low-density lipoproteins (LDLs), thereby contributing to the
The heme enzyme, myeloperoxidase (EC 1.11.1.7, MPO ),
is one of the key players in the first line of the nonspecific
1
immune defense system. In the presence of hydrogen per-
oxide and halide ions, MPO catalyzes the synthesis of hypo-
halous acids (e.g., hypochlorous acid, HOCl). These oxidizing
and halogenating compounds kill and destruct foreign micro-
organisms. MPO is packed in the azurophilic granules of
1
1
development of atherosclerosis. Indeed, LDLs oxidized by
MPO (Mox-LDL) can induce foam cell formation and trigger
1,12
inflammatory responses in monocytes and endothelial cells.
The interaction between MPO and LDL is facilitated by elec-
(
phagocytosing) polymorphonuclear leukocytes in relatively
highconcentrations (typically upto5%of cell dry weight) and
is released into the phagosomes that contain the endocytosed
pathogens. However, MPO can also be released outside the
1
trostatic interactions as MPO is strongly basic at physiolog-
ical pH, whereas LDLs are negatively charged. In addition, it
has been shown that MPO can oxidize the APO A1 of high-
density lipoproteins (HDLs), thus impairing reverse choles-
1
phagocytes and contribute to tissue damage at sites of
inflammation. In addition to its beneficial role in microbial
killing, MPO is reported to be involved in a growing number
of diseases because of its ability to generate reaction products
that can oxidize many types of biomolecule (lipids, proteins,
DNA, etc.).
MPO can influence disease pathogenesis in two ways: In the
2
first, MPO is directly involved at the origin of the disease; in
the second, MPO acts to worsen symptoms associated with
1
3,14
terol transport.
Because of these deleterious effects, devel-
opment of therapeutic strategies aimed at inhibiting MPO is
1
5
needed. However, despite several attempts, there are still no
molecules with sufficient in vitro and/or in vivo inhibiting
1
6-28
properties.
We recently assessed the MPO inhibiting effects of com-
,3
2
9
monly used anti-inflammatory drugs. Flufenamic acid (1,
Figure 1) was identified as a good candidate, which inhibited
29
both HOCl production and LDL oxidation. However, these
results were only observed at high concentrations, rendering
flufenamic acid unusable because of its toxic adverse effects at
these doses. Analogous compounds were also investigated but
†
The PDB code of MPO is 1DNW.
To whom correspondence should be addressed. Phone: 003226505235.
*
Fax: 003226505249. E-mail: dufrasne@ulb.ac.be. Address: Campus plaine,
CP 205/5, 1050 Brussels, Belgium.
a
Abbreviations: APO B-100, apolipoprotein; DIBAL-H, diisobutyl-
16
none had better inhibitory activity, likely because their
negative charge dampened the interaction between MPO and
aluminium hydride; DMA, dimethylacetamide; DMSO, dimethyl-
sulfoxide; EtOAc, ethyl acetate; EtOH, ethanol; LDL, low-density
lipoprotein; MPO, myeloperoxidase; TEA, triethylamine.
3
0
anionic LDLs. We, therefore, hypothesized that positively
r
2
010 American Chemical Society
Published on Web 11/22/2010 pubs.acs.org/jmc

8
748 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Soubhye et al.
Figure 1. General structure of flufenamic acid (1), tryptamine derivatives (2,3,4), and 5-fluoroindole-3-alkylamine derivatives (3).
charged compounds could overcome this problem and in
addition would allow more favorable interactions with anio-
nic amino acid residues at the active site of MPO.
Jantschko et al. (2005) showed that tryptamine (2) deriva-
tives act as substrates and inhibitors of MPO. Derivatives that
carry a metabolically stable substituent on position 5 of the
indole ring, such as chlorine (5-chlorotryptamine, 3) or fluo-
rine (5-fluorotryptamine, 4), were shown to be efficient rever-
3
1
sible inhibitors. It must be noted that the stability of the
aromatic moiety of potential inhibitors is crucial because the
indole ring is subject to oxidation by MPO, particularly on
3
2
position 5. Substitution by electron-withdrawing groups
makes the indole moiety very fragile and prone to cleavage
between C-2 and C-3. This has been demonstrated with
3
3,34
melatonin, which possesses an OMe group on position 5.
A promising MPO drug candidate must be a stable molecule
and good substrate so that administered doses and the risk of
adverse effects can be kept as small as possible.
By exploiting the positions of 5-fluorotryptamine obtained
from structure-based docking, we designed analogues that
could have additional interactions with the active site of the
heme enzyme. Two strategies were followed, namely varying
the length of the aminoalkyl side chain and adding substitu-
ents of different sizes to the amino group of the side chain.
These analogues were then synthesized and their capacity to
inhibit recombinant MPO, produced in CHO-cell lines, was
measured. We report the effect of these new compounds on
MPO-mediated taurine chlorination and LDL oxidation and
on the interaction with the “Compound I” and “Compound
II” redox intermediates of MPO. We also propose a mecha-
nism for the inhibition.
Figure 2. (A) Best pose of 5-fluorotryptamine (4) generated by the
Glide docking program. The ligand is depicted as stick. Neighbor-
ing residues including Thr100 and Glu102, which hydrogen bond to
the ligand and the heme (with carbon atoms in light gray), are also
illustrated. The small blue sphere indicates the location of the ferric
ion at the center of the heme. (B) View from the entrance of the
channel of the best pose of 5-fluorotryptamine (4) with a represen-
tation of the molecular surface of the protein and neighboring
hydrophobic, negatively charged residues and Thr100 (see text),
which were accounted for design the 5-fluorotryptamine analogues.
Results
Docking Experiments. Indole and tryptamine derivatives
have been reported to be reversible inhibitors of human
MPO with 5-fluorotryptamine being the most effective.
These findings prompted us to perform a rational structure-
based design of new tryptamine derivatives based on the
docking of 5-fluorotryptamine. The reliability of the docking
procedure was previously assessed by comparing the docking
of salicylhydroxamic acid with its position in the crystal
3
1
1
6
structure of the MPO complex.
be exploited to create structural modifications of 5-fluoro-
tryptamine (Figure 2B). In particular, the voids facing
Phe99, Phe147, Leu420, Phe407, and Leu406 could be filled
with chemical groups so as to form additional hydrophobic
interactions between the ligand and the binding site. On the
basis of these observations, we developed two strategies for
creating the analogues: (i) varying the length of the alkyl side
chain and (ii) adding different alkyl or cyclic substituents on
the side chain nitrogen. The amino group of 5-fluorotrypt-
amine that bears a positive charge at physiological pH was
conserved so as to keep the potential formation of an ionic
The best pose of 5-fluorotryptamine (Figure 2A) featured
stacking of the indole 6-membered ring onto the pyrrole ring
D of the heme. Stacking was also observed in the crystal
structure of the MPO structure in complex with salicylhy-
droxamic acid. In addition to the stacking, one salt bridge
was formed with Glu102 and one hydrogen bond with
Thr100. No hydrogen bond was observed between the indole
NH group and the protein. Another pose featured a hydro-
gen bond but at the expense of the stacking. Interestingly,
empty pockets remained around the docked ligand and could

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8749
Table 1. IC50 Values for the Inhibition of MPO, Predicted Free Energies
of Binding Obtained from Docking Experiments
a
no.
n
R
1
R
2
IC50 (μM)
ΔG (kcal/mol)
6
7
8
4
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
4
5
6
H
H
0.9 ( 0.3
0.2 ( 0.2
1.0 ( 0.1
-5.3
-6.3
-7.3
-6.5
-6.6
-6.8
-6.6
-6.5
-7.0
-6.3
-6.8
-7.7
-6.4
-6.7
-6.6
-7.5
-6.8
-5.7
-7.0
-7.5
-8.5
-6.6
-6.4
-5.9
C
N-methylpiperazinyl
2
H
5
C
2
H
5
H
CH
H
0.20 ( 0.03
0.20 ( 0.02
0.3 ( 0.1
0.80 ( 0.02
1.03 ( 0.08
0.09 ( 0.06
0.16 ( 0.08
0.04 ( 0.03
0.2 ( 0.1
0.050 ( 0.008
0.2 ( 0.2
0.3 ( 0.1
0.17 ( 0.08
1.5 ( 0.5
0.13 ( 0.09
0.35 ( 0.09
0.32 ( 0.01
0.35 ( 0.06
0.015 ( 0.004
0.008 ( 0.002
0.26 ( 0.01
16
17
18
19
20
21
22
23
15
24
25
26
27
28
29
30
31
34
41
48
3
H
C
C
C
2
3
4
H
H
H
5
7
9
H
H
H
CH
3
CH
3
C
2
H
5
2 5
C H
pyrrolidinyl
N-methylpiperazinyl
H
CH
H
3
H
C
C
C
2
3
4
H
H
H
5
7
9
H
H
H
CH
CH
C
3
3
2
H
5
2 5
C H
pyrrolidinyl
N-methylpiperazinyl
H
H
H
H
H
H
Figure 3. (A) Docking poses of compound 34 illustrating the
stacking with pyrrole D of the heme and the ionic interaction with
Glu102. (B) Docking poses of compound 6 and 34 illustrating the
absence of ionic interaction and the poor stacking of compound 6
compared to compound 34. The poses are generated by the Glide
docking program. Compounds 6 and 34 are depicted as green and
grey sticks respectively. The small blue sphere indicates the location
of the ferric ion at the center of the heme.
a
Compounds are classified first following the length of the side chain
and the substituents, then, respectively, according the mono- or disub-
stitution of the amino group. The IC50 value is the mean ( SD of 3
independent experiments.
interaction with Glu102 and/or the propionic heme group or
even to reach further negatively charged residues, such as
Glu116, Asp214, or the C-terminus of Ala104 (see Figure 2B).
The potential binding modes of these designed compounds
were analyzed by docking.
Side Chain Length Variations. The number of carbons in
the alkyl side chain of 5-fluorotryptamine (compound 4) was
modified. Compound 34, with a 4-carbon side chain length,
was predicted to have the same affinity as the compounds
that had 2, 3, and 5 carbon atoms (4, 15, and 41). Its predicted
affinity was 1.3 kcal/mol greater than that of the 1-carbon-
chain compound (compound 6) (see Table 1). The poses
found for compound 34 showed a stacking arrangement
with the pyrrole ring D of the heme and the formation of two
salt bridges with Glu102 and the heme propionate group
indole NH hydrogen bonds to the propionic group were
maintained but no salt bridge was formed.
Amino Group Side Chain Substituents. In another attempt
to improve the potency of 5-fluorotryptamine, the effect of
adding different substituents to the side chain amino group was
analyzed. Compounds with linear or branched alkyl group
substitutions on the side chain nitrogen had predicted scoring
values close to those of unsubstituted compounds (see Table 1).
Compound 26 had a score 1 kcal/mol more favorable than
5-fluorotryptamine (4). Its best pose showed hydrogen bonding
to the propionic acid group and formation of a salt bridge with
Glu102. Hydrophobic contacts mainly involved Phe99, Pro145,
Phe146, Phe147, Phe407, Leu415, and Leu420.
(
Figure 3A). Compared to 5-fluorotryptamine binding, the
We also probed cyclic groups as substituents on the amino
group of the side chain in order to introduce new interactions
and to reduce or avoid the energy penalty for immobilization of
rotatable bonds upon binding of alkyl substituents. Two frag-
ments were used: Pyrrolidinyl and N-methylpiperazinyl moieties.
Compound 31 had the most favorable energy (-8.5 kcal/mol).
The docked positions of this compound featured stacking with
the heme pyrrole ring D, one hydrogen bond with the propionic
group, an electrostatic interaction with Glu102 (not a salt bridge),
and hydrophobic contacts with Phe407.
indole moiety penetrated deeper into the pocket on top of the
heme and the fluorine atom was closer to the heme iron.
There were several hydrophobic contacts with Phe99 and
with the aliphatic chain of one of the heme propionates. With
the 1-carbon-chain (6), there was less stacking and one salt
bridge was lost but one additional hydrogen bond was formed
between the indole NH group and the heme (Figure 3B). The
fluorine atom was not oriented toward the iron atom. The
compound bearing a 6-carbon side chain (48) featured poses
in which either the stacking was lost and the side chain
nitrogen made a salt bridge with Glu116 located at the
entrance of the catalytic pocket or shifted stacking and the
The analysis of the binding modes predicted by docking of
these analogues and their binding energy values encouraged
us to synthesize them for further evaluation.

8
750 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Soubhye et al.
a
Scheme 1. Synthesis of Compound 6
a
Reagents and conditions (i) NH
2
OH.HCl, pyridine; (ii) H
2
, Pd/C, HCl, EtOH.
a
Scheme 2. Synthesis of Substituted 3-(Aminomethyl)indoles
a
Reagents and conditions: (i) formaldehyde (37 wt % aqueous solution), acetic acid, dioxane.
a
Scheme 3. Synthesis of the Intermediate Alcohols and Unsubstituted 3-(Aminoalkyl)indoles n = 2 and 3 (4 and 15)
a
Reagents and conditions: (i) dihydrofuran or dihydropyran, H
Pd/C 10%, EtOH.
2 4 2 2 3 2
SO , DMA; (ii) methanesulfonyl chloride, TEA, CH Cl ; (iii) NaN , DMSO; (iv) H ,
Chemistry. Compound 6 was prepared as indicated in
Scheme 1. The carbonyl group of the commercially available
(for example, 95% versus 72% for the synthesis of 9). The
starting alcohols 9 and 10 were transformed into their mesylate
derivatives in good yields by adding methanesulfonyl chloride at
room temperature in the presence of triethylamine. Mesylate
groups were then converted into azidoalkanes to give com-
pounds 13 and 14, which were hydrogenated using Pd/C as
5
corresponding oxime to give compound 5. Hydrogenation
-fluoroindole-3-carboxaldehyde was transformed into the
3
5
of the latter, catalyzed by Pd/C 10% in the presence of HCl,
3
led to compound 6.
6
39
For the series of indoles substituted by aminomethyl moieties,
aminoalkylations of commercially available 5-fluoroindole
were performed using the Mannich reaction, i.e., in the pres-
ence of secondary amines and formaldehyde, resulting in the
catalyst to create compounds 4 and 15 (Scheme 3).
To synthesize compounds 16 to 31, which were substituted
on the aliphatic nitrogen atom, mesylate derivatives 11 and
12 were added to the corresponding amines in dioxane at
3
7
40
synthesis of compounds 7 and 8 (Scheme 2).
100 ꢀC (Scheme 4).
To obtain 3-(4-aminobutyl)-5-fluoro-1H-indole oxalate
For the syntheses starting from 5-fluoro-3-(hydroxyalkyl)-
indoles, a general procedure based on the Fisher indole syn-
thesis was applied using 4-fluorophenylhydrazine hydrochloride
and dihydrofuran or dihydropyran for the ring formation step.
To improve the results for this step, we performed the reac-
41
34, the mesylate group in 12 was converted to nitrile 32,
which was further hydrolyzed by KOH in t-BuOH to give
4
carboxamide 33. Reduction of the amide using LiAlH in
2
4
4
3
dioxane led to the desired amine 34 (Scheme 5).
The synthesis of compound 41 followed the same syn-
thetic path as for the indole derivative 34 but started from
3
8
tion described by Campos and colleagues under microwave.
These conditions led to shorter reaction times and higher yields

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8751
a
Scheme 4. Synthesis of Compounds 16-31
a
Reagents and conditions: (i) dioxane, 100 ꢀC.
a
Scheme 5. Synthesis of Compound 34
a
Reagents and conditions: (i) NaCN, H
2
O, DMA, 100 ꢀC; (ii) KOH, t-BuOH; (iii) LiAlH , dioxane, 100 ꢀC.
4
a
Scheme 6. Synthesis of Compound 41
a
Reagents and conditions: (i) DIBAL-H, CH
2
Cl
2
, -78 ꢀC; (ii) 4-fluorophenylhydrazine hydrochloride, H
, dioxane, 100 ꢀC.
2 4
SO , DMA; (iii) methanesulfonyl chloride,
TEA, CH
Cl ; (iv) NaCN, H
2 2
2
O, DMA, 100 ꢀC; (v) KOH, t-BuOH; (vi) LiAlH
4
6
-hydroxyhexanal 36, which was obtained by the reduction
side chain on position 3 of 5-fluoroindole, as well as by the
alkyl groups substituting its amino group. Compound 41
with a 5-carbon nonsubstituted side chain was the most
efficient inhibitor. Lengthening the side chain from n=1
to n=5 increased the inhibitory potency. With the exception
of substitution by dimethyl or pyrrolidinyl groups, substitu-
tion of the side chain nitrogen did not improve the inhibition.
Compound 27 with a 3-carbon chain and a butyl substituent
displayed the weakest activity. A closer analysis of com-
pounds that had the same side chain length but different
substituents revealed striking features. Among the molecules
with a 2-carbon side chain, compound 22 with a pyrrolidine
and compound 20 with a dimethylamino group exhibited the
greatest activity. In this series, the size of the alkyl substit-
uents (except for very bulky groups) apparently did not
affect the inhibitory potency (compound 19). In the 3-carbon
side chain series, the unsubstituted indole 15 had the most
inhibitory activity. In this series, the volume of the substit-
uents did not significantly change the interaction with MPO
(as for 2-carbon side chain molecules). Finally, among all the
fluorotryptamine derivatives, the 1-carbon chain compounds
displayed marginal inhibitory activities.
44
of ε-caprolactone 35 using DIBAL-H (Scheme 6). The al-
dehyde was then subjected to a Fisher reaction with 4-fluoro-
phenylhydrazine hydrochloride, giving the alcohol 37, which
was further converted into the mesylate 38. A nucleophilic
substitution was performed on compound 38 using NaCN,
and the resulting nitrile 39 was then hydrolyzed into the
amide 40. After reduction of the latter by LiAlH , the final
4
compound (41) was isolated as its oxalate salt.
Finally, the fluorotryptamine derivative with 6 methylene
groups in the side chain was obtained, starting from cyclo-
octanone 42 after a Baeyer-Villiger rearrangement and reduc-
4
5
tion of the resulting lactone 43 into aldehyde 44 (Scheme 7).
The usual synthetic scheme was then used, as for compound
4
synthesis.
Biological Activity. A microplate reader was used to moni-
tor inhibition of MPO-mediated taurine chlorination in a
4
6
high throughput screening mode. Table 1 lists the IC50
values of the 3-(aminoalkyl)-5-fluoroindole analogues. The
presented IC50 values clearly demonstrate that the capacity
of the investigated compounds to inhibit MPO-mediated
chlorination was modulated by the length of the aminoalkyl

8
752 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Soubhye et al.
a
Scheme 7. Synthesis of Compound 48
a
Reagents and conditions: (i) m-chloroperbenzoic acid, CH
2
Cl
Cl ; (v) NaN
2
, 70 ꢀC; (ii) DIBAL-H, CH
2
Cl
2
, -78 ꢀC; (iii) 4-fluorophenylhydrazine hydrochloride,
H
2
SO
4
4%, DMA; (iv) methanesulfonyl chloride, TEA, CH
2
2
3 2
, DMSO, 100 ꢀC; (vi) H , Pd/C 10%, EtOH.
Table 2. IC50 Values for the Inhibition of the Oxidation of LDL Carried
Out by MPO/Cl /H O
2 2
chain had less inhibitory potency and compounds with a
-
1
-carbon side chain were weak inhibitors of LDL oxidation.
Two compounds, however, attracted attention. Compound
7 had a poor inhibitory effect on MPO-mediated taurine
2
chlorination but was an excellent inhibitor of LDL oxida-
tion. In contrast, compound 22 had a small effect on LDL
oxidation (except at the higher concentration) despite a
marked effect on the chlorination activity of MPO.
no.
n
R
1
R
2
IC50 (μM)
Two redox intermediates are relevant in the enzymology of
MPO, namely Compound I and Compound II. Because
tryptamine derivatives have been shown to act as reversible
a
6
7
8
4
1
1
1
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
4
5
6
H
H
>1
a
C
2
H
5
C
2
H
5
>1
a
31
N-methylpiperazinyl
H
CH
>1
inhibitors and interact with both redox intermediates, it
a
H
H
H
H
H
0.94
was important to also study the direct interaction of the most
efficient inhibitors (compounds 34 and 41) with Compound I
and Compound II by sequential-mixing stopped-flow spec-
a
1
1
1
1
2
2
2
2
1
2
2
2
2
2
2
3
3
3
4
4
6
7
8
9
0
1
2
3
5
4
5
6
7
8
9
0
1
4
1
8
3
0.65
a
C
C
C
2
3
4
H
H
H
5
7
9
0.85
a
0.46
47
a
troscopy (Figures 5 and 6). Compound I is formed by the
0.25
a
reaction of hydrogen peroxide with the ferric state of MPO
(Figure 4). Thereby, the O-O bond is cleaved heterolyti-
cally, forming a redox intermediate called Compound I. In
this fast reaction, Fe(III) is oxidized to an oxoferryl species
[Fe(IV)dO] and the porphyrin to the corresponding
π-cation radical (reaction 1 in Figure 4). Compound I is
deficient in two electrons and restoration of the ferric state
can be accomplished by either direct two-electron reduction
CH
3
CH
3
0.58
a
C
2
H
5
C
2
H
5
0.51
a
pyrrolidinyl
N-methylpiperazinyl
1.03
a
0.65
a
H
CH
H
0.65
0.026
b
3
H
a
C
C
C
2
3
4
H
H
H
5
7
9
H
0.19
a
H
0.34
0.017
b
H
-
-
by halides (Cl or Br ), thereby forming the corresponding
hypohalous acids (reaction 2 in Figure 4), or by two one-
electron reduction steps mediated by aromatic electron
donors (including indole derivatives) via Compound II
b
CH
C
3
CH
C
3
0.049
a
0.39
2
H
5
2
H
5
b
pyrrolidinyl
N-methylpiperazinyl
0.21
a
0.31
0.012
4
9
b
[
Fe(IV)-OH] (reactions 3 and 4 in Figure 4). The halogena-
H
H
H
H
H
H
b
0.005
a
0.58
tion cycle includes reactions 1 and 2, whereas the peroxidase
cycle includes reactions 1, 3, and 4. It is important to note
that Compound II cannot oxidize chloride or bromide.
Figures 5 and 6 demonstrate the kinetics of the reactions
between compound 34 and MPO Compound I and Com-
pound II. Very similar time traces were observed with
compound 41 (not shown). The inhibitors acted as one-
electron donors to both redox intermediates of MPO. Fig-
ure 5 depicts the direct transition of Compound I to Com-
pound II mediated by oxidation of compound 34. The
reaction was monophasic (Figure 5B), and from the linear
dependence of kobs values on substrate concentration, ap-
a
IC50 values calculated by measuring the inhibition of the LDL
oxidation at three concentrations (1000, 100, and 50 nM; see Supporting
b
Information). IC50 values were calculated from sigmoid curves ob-
tained by measuring the inhibition with seven concentrations (5.0, 7.5,
10.0, 25.0, 50.0, 500.0, 1000.0 nM).
To measure MPO-dependent LDL oxidation, which is a
contributing factor in atherogenesis, an ELISA was recently
developed, based on a mouse monoclonal antibody (Mab
AG9) that specifically recognizes MPO-oxidized APO B-100
3
0
7
-1 -1
on LDL. Table 2 lists the IC50 values for the inhibition of
LDL oxidation by the different compounds. Most of the
compounds described in this study effectively inhibited LDL
oxidation at submicromolar concentrations. Compounds 34
and 41 and, to a lesser extent, 24 and 27, showed the best
inhibition profile. Other compounds with a 3-carbon side
chain also exhibited rather good inhibitory activity (except
compound 15). For comparison, indoles with a 2-carbon side
parent bimolecular rates (k ) of (1.1 ( 0.1) Â 10 M
s
(compound
3
7
-1 -1
(compound 34) and (1.0 ( 0.1) Â 10 M
s
41) were calculated (Figure 5C). The high intercept clearly
demonstrated that Compound II was not stable but further
transformed. This is highlighted by Figures 6A and 6B that
show the direct monophasic transition of Compound II to
native MPO mediated by compound 34 with a clear isosbestic
point. The apparent bimolecular rate constants (k ) were
4

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8753
Figure 4. Scheme of chlorination cycle and peroxidase cycle of myeloperoxidase (MPO). Architecture of heme and conserved distal residues
His95 and Arg239 was obtained from X-ray structure (PDB: 1CXP). At the heme edge (δ-meso bridge between pyrrol rings A and D),
substrates bind and donate electrons. In reaction (1): ferric MPO is oxidized by hydrogen peroxide to Compound I [i.e., oxoiron(IV) porphyrin
radical cation]. In reaction (2), Compound I is directly reduced back to the resting state by chloride (or bromide) thereby releasing
hypochlorous (or hypobromous) acid. Reactions (1) and (2) constitute the halogenation cycle. In reaction (3), Compound I is reduced to
Compound II [i.e., protonated oxoiron(IV)] by a one-electron donor (e.g., an indole derivative). In reaction (4), Compound II is reduced to the
native state thereby oxidizing a second substrate molecule. Reactions (1), (3), and (4) constitute the peroxidase cycle. Note that Compound II
cannot oxidize chloride or bromide.
-
1
-1
-1 -1
3
(
60 M
s
(compound 34) and 506 M
s
(compound 41)
Figure 6C). The k /k ratios of 30555 and 19762 suggest that
poses provide a tentative explanation for the high IC50 value
for the 1-carbon side chain; because of the short alkyl chain,
the nitrogen group cannot reach the negatively charged
Glu102 to form a salt bridge, as observed for the 2-, 3-, 4-,
and 5-carbon side chains. However, a further increase to a
3
4
Compound II reduction was the rate limiting step in the per-
oxidase cycle. Both inhibitors were excellent electron donors for
Compound I, but poor substrates for Compound II.
6
-carbon side chain again increased the IC .
50
In contrast, in 2- and 3-carbon side chain compounds, the
Discussion and Conclusions
inhibitory potencydecreasedwhenthe side chainaminogroup
was substituted with linear alkyl substituents of increasing
length. In most cases, compounds with an N-substituted side
chain showed higher IC50 values compared to unsubstituted
compounds with the same side chain length. However, com-
pounds with two substituents had more favorable inhibitory
properties compared to those with just one linear substituent
(e.g., compare compound 19 with compound 21 or compound
27 with compound 29). The docked poses suggest that disub-
stituted amino groups fit better into the hydrophobic pocket
of MPO.
Compounds with cyclic substituents were not potent in-
hibitors, with the exception of compound 22. The experimen-
tal data and the docked binding modes suggest that the steric
hindrance on the side chain amino group and the flexibility of
the substituents may be important factors for the structure-
activity relationship.
We used structure-based docking of 5-fluorotryptamine (4)
in the active site of MPO to design derivatives with potent
inhibitory action against MPO (see Table 1 and 2). All the de-
signed derivatives could be docked into the active site pocket.
The best poses featured stacking between the indole and the
pyrrole moieties although this interaction was not ideal in
all cases. The docking energy binding values of these com-
pounds ranged between -5.3 and -8.5 kcal/mol with a value
of -6.5 kcal/mol for 5-fluorotryptamine (4). Given the diffi-
culty in evaluating affinity using the scoring functions devel-
oped for the docking programs, we could not reliably dis-
criminate between the different ligands, and as a result, all the
designed compounds were synthesized and their IC50 values
were measured.
In unsubstituted compounds, the distance between the
indole moiety and the side chain nitrogen atom appeared to
be an important parameter for binding to, and inhibition of,
MPO. Lengthening the side chain from 1 to 5 carbons increased
the inhibitory potency by 2 orders of magnitude. The docked
There was no clear correlation between the calculated dock-
ing binding energies and the IC50 values derived from the two in
vitro inhibition assays. This illustrates that molecular docking

8
754 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Soubhye et al.
Figure 5. Reaction of MPO Compound I with compound 34 (pH 7.4 and 25 ꢀC). (A) Spectral changes upon reaction of 2 μM MPO Compound
I with 10 μM compound 34. First spectrum after mixing corresponds to Compound I subsequent spectra were recorded after 1.3, 3.8, 6.4, 8.9,
1
(
1.5, 14.1, and 16.6 ms. Arrows indicate spectral changes. (B) Typical time trace and fit of MPO Compound II formation followed at 456 nm.
C) Pseudo-first-order rate constants for MPO Compound I reduction plotted against compound 34 concentration.
maybehelpful in predicting bindingmodes of a molecule to an
active site but cannot easily be used for ranking the inhibitory
capacity. In the present study, the inhibiting molecules were
also substrates and their efficiency to interact with MPO
Compounds I and II depended also on the redox potential
of the bound molecule itself, which is not necessarily corre-
lated with the binding affinity. As a result of the poor scoring
correlation, “induced-fit” studies were undertaken for two com-
pounds selected as the least and most potent inhibitors (27 and
corroborated by the inhibition of taurine chlorination. A
relationship between inhibition of taurine chlorination and
of LDL-oxidation is obvious for the most potent inhibitors.
This demonstrates that these different molecules were able to
inhibit LDL oxidation in spite of the binding between MPO
48
and LDL. Binding of MPO to LDL during LDL oxidation
is responsible for the oxidative modification of APO B-100 in
plasma. However, this event could also block the catalytic site
of the enzyme and, therefore, interfere with enzymatic inhibi-
1
6
34). Docked poses differed only by the positions of their side
tion by providing some protective effect. The heme group of
MPO that is responsible for the enzymatic activity is indeed
located in a distal hydrophobic cavity with a narrow oval-
chains and the scored affinity shifted from -6.8 to -5.8 and
from -6.6 to -7.6 kcal/mol for compounds 27 and 34, respec-
tively. This observation suggests that “induced-fit” effects could
be important for binding of fluorotryptamine analogues.
Inhibition parameters for LDL oxidation are relevant
because they reflect the potential in vivo effects of the com-
pounds (see Table 2). One of the most striking observations is
that substitution by N-methylpiperazinyl in the compounds
with 2-carbon side chains increased the inhibitory potency in
comparison with structurally closely related compounds in
terms of chain length and steric hindrance (for example,
compare compounds 22 and 23). Compound 23 was the
weakest in its series in the MPO chlorination inhibition assay
3
0
shaped opening. This characteristic might explain the ob-
served differences in the inhibition of MPO-mediated taurine
chlorination and LDL oxidation. As far as binding to LDL is
concerned, the lipophilicity of potent inhibitors seems to be an
important factor. Indeed, the formation of a complex macro-
molecular structure between MPO and LDL, mainly through
electrostatic interactions, may modify the chemical and steric
requirements for good penetration of substrates and/or in-
hibitors into the heme cavity. Parameters such as charge and
lipophilicity of compounds have already been evoked to
1
6,30
explain similar results.
(
see Table 1) but had a very good activity in the inhibition of
The transient kinetics study demonstrated that the probed
compounds act as electron donors of both relevant redox inter-
mediates of MPO, namely Compound I and Compound II.
Detailed kinetic parameters could be elucidated for the most
potent inhibitors, compounds 34 and 41. Substrates for MPO
(halides or aromatic compounds) have been demonstrated to
LDL oxidation (see Table 2). The reason for this sudden
increase in LDL oxidation inhibitory activity for compound
23 is unknown and might be ascribed to the presence of a
second nitrogen. The second important observation is the
very marked activity of compounds 34 and 41, which was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8755
Figure 6. Reaction of MPO Compound II with compound 34 (pH 7.4 and 25 ꢀC). (A) Spectral changes upon reaction of 2 μM MPO
Compound I with 500 μM compound 34. First spectrum after mixing corresponds to Compound II, subsequent spectra were recorded after 2.3,
4
4
.7, 8.36, 11, 14.6, 19.2, and 44.1 s. Arrows indicate spectral changes. (B) Typical time trace and fit of MPO Compound II reduction followed at
56 nm. (C) Pseudo-first-order rate constants for MPO Compound II reduction plotted against compound 34 concentration.
bind and deliver electrons at the entrance to the distal heme
pocket near the δ-meso bridge (between pyrrol ring A and D;
human heme peroxidases cannot oxidize these inhibitors. As a
result, the new tryptamine analogues decrease the pool of
native MPO and Compound I responsible for the oxidative
damage and do not interfere with homologous peroxidases.
In conclusion, a series of 3-(aminoalkyl)-5-fluoroindole
analogues was designed using structure-based docking of
4
9,50
see Figure 4),
which is supported by the presented docking
data. It is reasonable to assume that substrate binding sites at
Compounds I and II are identical. This assumption is based
on the fact that structural differences in the heme pocket even
between (high-spin) ferric MPO and its (low-spin) cyanide
5
-fluorotryptamine. These compounds were synthesized and
5
0
assessed in vitro. Some of these compounds showed consider-
able potency for inhibition of taurine chlorination and of
LDL oxidation mediated by MPO. The more potent mole-
cules possess a 4- or 5-carbon length side chain and no sub-
stituent on the side chain nitrogen. These are the first mole-
cules that have been shown to inhibit LDL oxidation in vitro
at nanomolar concentrations, thus providing an excellent
basis for further development of drugs targeting MPO. The
discrepancies observed in the different MPO inhibition assays
complex are negligible. The cyanide complex of a perox-
idase is a suitable model for the (low-spin) oxoferryl-state of
5
Compound I and Compound II. Consequently, the ob-
0
served differences between the apparent reaction rates, k3
and k , must be the result of differences in the redox
4
properties of Compound I and Compound II. Because of
the very high reduction potential of MPO Compound I, both
inhibitors were rapidly oxidized, thereby directly reducing
4
9
Compound I to Compound II. Compared to Compound I,
Compound II is a weaker oxidant, as reflected by high k /k
(Table 1 and Table 2) provide insight into the interaction
3
4
between MPO and LDLs during LDL oxidation, which can
be further studied in in vivo experiments.
ratios. Similar findings have been reported for 5-fluorotryp-
tamine, suggesting that these compounds act as reversible
3
1
inhibitors. Because of their marked efficiency in one-elec-
tron reduction of Compound I, they shift MPO from the
chlorination cycle to the peroxidase cycle (Figure 4) because
Compound II is not able to oxidize chloride or bromide. The
MPO-typical high difference in reduction potentials between
Compounds I and II (which is not observed in homologous
lactoperoxidase or eosinophil peroxidase) guarantees that a
high percentage of MPO stays as Compound II and that other
Experimental Section
Docking Experiments. Protein and Ligand Preparation. The
X-ray structure of human myeloperoxidase complexed to cya-
nide and thiocyanate (PDB code: 1DNW) was used as the target
50
structure to endeavor the docking studies. The X-ray water
-
-
and CN and SCN molecules were removed from the active
site. The ligand input files were prepared according to the
following procedure. The initial 3D structures of the ligands

8
756 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Soubhye et al.
5
1
were generated using Corina, and the ligand partial charges
were ascribed using the OPLS force-field as performed by
General Procedures for the Synthesis of Compounds 7 and 8.
To a solution of 5-fluoroindole (200 mg, 1.6 mmol) in dioxane
(5 mL) was added a mixture of the amine (1.6 mmol) and for-
maldehyde (145 μL, 37 wt % solution in water) in dioxane/acetic
acid 1:1 (6 mL). The resulting mixture was stirred at room
temperature overnight and the solvent was evaporated under
reduced pressure. To the residue, a solution of 0.1 M KOH (30 mL)
and EtOAc were added. Then the organic layer was washed with
water, dried with Na SO , and evaporated under vacuo. The
52
Glide. As part of an effective 3D ligand structure preparation
process the Epik program was used to predict pK values and
generate protonation states of all ligands. All ligands feature
pK values higher than 8.5 for their side chain nitrogen. They
a
were thus docked in their protonated form.
Docking Procedure. The Glide (Grid-Based Ligand Docking
With Energetics) algorithm approximates a systematic search of
positions, orientations, and conformations of the ligand in the
receptor binding site using a series of hierarchical filters (www.
schr o€ dinger.com). In the present work, the binding region was
a
5
3
2
4
residue was dissolved in diethylether and extracted with 0.1 M
HCl. The aqueous layer was rendered alkaline with 1 M KOH
(pH ≈ 10) and extracted with diethylether. The organic layer
˚
˚
˚
defined by a 18 A Â 18 A Â 18 A box centered on the central
position of the cyanide in the crystal complex. At most 10 poses
were generated for each molecule. The protein structure was
prepared using the Protein Preparation Wizard in the Schr o€ dinger
software graphical user interface Maestro. Glide XP docking
protocol and scoring function were used to score the poses of
the different ligands. The default settings of Glide version 4.5
were used for the remaining parameters. Induced-fit protocols
were performed for compound 34 and 27. Initial Glide docked
poses were obtained in binding sites with Phe99, Glu102, Arg239,
and Phe407 mutated into alanine. Side-chain and backbone mini-
mizations as well as docked poses refinement were carried out in
structures with the alanine side chains replaced by their wild type
corresponding residues. Glide redockings were carried out in
these minimized structures.
Synthesis. H and C NMR spectra were taken on a Bruker
Avance 300 spectrometer at 293 K (Bruker, Wissembourg,
France). δ are given in ppm relative to TMS, and the coupling
constants are expressed in Hz (frequencies for H and C:
respectively 300 and 75 MHz). IR analyses were performed on
a Shimadzu IR-470 spectrophotometer and the peaks data are
given in cm . The mass spectra were obtained on QTOF 6520
Agilent, Palo Alto, CA, USA), positive mode, ESI, mode TOF,
by diffusion of 0.5 mL/min, with mobile phase HCOOH 1%:
CH
OH (50:50), (VCAP 3500 eV; Source Tꢀ, 350 ꢀC; fragmen-
tor, 110 V; Skinmer, 65 V). All reactions were followed by TLC
carried out on Fluka PET-foils Silica Gel 60 and compounds
were visualized by UV and by spraying Van Urk reagent (0.13 g
of p-dimethylaminobenzaldehyde dissolved in a mixture of
was dried on Na SO and evaporated
2 4
General Procedure for the Synthesis of Compounds 9, 10, 37,
3
8
and 45. A solution of 4-fluorophenylhydrazine hydrochloride
(1 g, 6.9 mmol) in 4 wt % aqueous H SO (10 mL) and DMA
2
4
(10 mL) was heated at 100 ꢀC under microwave. To this solution
was added dihydrofuran, dihydropyran, or the corresponding
hydroxyaldehyde (6.9 mmol) dropwise over 2 min. Then the
reaction was heated at 175 ꢀC (temperature ramp from room
temperature to 100 ꢀC over 8 min, then addition of the reagent,
heating to 175 ꢀC over 8 min and maintaining this temperature for
30 min, 1000 W). The reaction mixture was cooled to room
temperature and extracted two times with EtOAc (25 mL), and
the organic layer was washed with water and evaporated. The
crude material was purified by column chromatography (eluent
CH Cl /EtOAc 4:1).
2
2
1
13
General Procedure for the Synthesis of Compounds 11, 12, 38,
5
4
and 46. A solution of alcohol (5.5 mmol) in CH
was cooled with an ice bath. To this solution TEA (0.44 mL,
.8 mmol) and methanesulfonyl chloride (0.77 mL, 5.5 mmol)
were added. The solution was stirred at room temperature for
h. Then it was washed with a saturated solution of NaHCO
and H O and then dried over Na SO and filtered. The solvent
2 2
Cl (25 mL)
1
13
5
2
3
-
1
2
2
4
was evaporated under reduced pressure. The crude material was
purified by flash chromatography (CH Cl /EtOAc 4:1).
General Procedure for the Synthesis of Compounds 13, 14, and
(
2
2
3
5
5
4
7. To a solution of sulfonate (3.6 mmol) in DMSO (20 mL),
NaN (3.6 mmol) was added, and the solution was stirred at
room temperature for 4 h (compounds 13 and 14) or for 1 h at
00 ꢀC (compound 47) . Then H O (30 mL) and toluene (30 mL)
were added. After decantation, the organic layer was washed
with water, dried with Na SO , filtered, and evaporated under
reduced pressure. The residue was purified by column chroma-
tography (CH Cl ).
3
1
2
1
00 mL of 65 wt % H SO and 0.1 mL of 5 wt % FeCl ). Column
2 4 3
chromatographies were performed with EchoChrom MP silica
3-200 from MP Biomedicals. The starting compounds 4-phe-
nylhydrazine hydrochloride, dihydrofuran, dihydropyran, and
-fluoroindole were available from Sigma-Aldrich. Starting
compound 5-fluoroindole-3-carboxaldehyde was purchased
from Alfa Aesar. Reactions under microwave were performed
with a Start-S microwave oven from Milestone (Milestone, Sorisole,
Italy). Purity was determined with LC-DAD (Waters, Milfors,
USA) using a 150 mm  4.6 mm Symmetry C18 column at a
mobile phase flow rate of 1 mL/min. The mobile phase was a
mixture of methanol (350 mL) and a KH PO solution (0.07 M
2
4
6
2
2
General Procedure for the Synthesis of Compounds 16-31. A
solution of mesylate 11 or 12 (3.6 mmol) in dioxane (5 mL) was
added slowly through an addition funnel to a refluxing solution
of the amine (0.26 mol) in dioxane (15 mL) at 100 ꢀC. After the
addition was completed, the reaction medium was stirred at this
temperature for 4 h. After cooling, the mixture was treated with
water (20 mL) and extracted with EtOAc (30 mL). The organic
5
2 4
layer was dried over Na SO and evaporated to dryness to
2
4
in water, 650 mL) adjusted to pH 3.0 with a 34 wt % H PO
3
afford a crude product. The residue was dissolved in 0.1 M
HCl. This solution was washed with diethylether and rendered
alkaline (pH ≈ 10) with a solution of 1 M NaOH. The mixture
was extracted with ether. The organic layer was washed with
4
solution. The chromatograms were extracted at maximum
absorption wavelengths by using the Max Plot extraction mode.
The purity was g95% for all the compounds.
General Procedures. General Procedures for the Synthesis of
Compounds 4, 15, and 48. Palladium on charcoal 10% (50 mg)
was added to a solution of azide derivative (4.6 mmol) in EtOH
2 4
water, dried over Na SO , and evaporated to afford the crude
base as a solid. When bases were obtained as liquids, corre-
sponding tartaric acid salts were prepared according to the
following method. After evaporation of ether, the residue was
taken up with a hot solution of D-tartaric acid (1 equivalent) in
2-propanol (10 mL). The mixture was cooled and the formed
solid was filtered and dried under vacuum.
(
20 mL). The suspension was stirred under H (4.1 bar) overnight.
2
After filtration on Celite, the solvent was evaporated under re-
duced pressure. The residue was dissolved in ether, extracted with
0.1 M HCl, and the resulting solution was washed with ether. A
solution of 1 M NaOH was added (pH ≈ 10), and the mixture was
General Procedure for the Synthesis of Compounds 32 and 39.
To a solution of NaCN (2 g, 41 mmol) in H O (10 mL), DMA
2
(10 mL) was added and heated at 100 ꢀC. Then, a solution of the
mesylate (7.4 mmol) in DMA (10 mL) was added dropwise. The
reaction was stirred at this temperature for 2 h and then cooled to
room temperature and extracted with EtOAc. The organic layer
extracted with diethylether. The organic layer was washed with
water, dried over Na SO , and evaporated under reduced pressure.
2
4
For compound 4, a saturated solution of anhydrous oxalic acid in
ether was added to the ethereal solution and the resulting solid was
filtered, washed with ether, and dried under vacuum.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8757
was washed with water, dried on Na SO
2
4
, and evaporated. The
residue was purified by flash chromatography (CH Cl /EtOAc 4:1).
Inhibition of LDL Oxidation. LDL oxidation was carried
out at 37 ꢀC in a final volume of 500 μL. The reaction mixture
contained the following reagents at the final concentrations
indicated between brackets: pH 7.2, PBS buffer, MPO (1 μg/mL),
LDL (1000 μg/mL), 2 μL 1 N HCl (4 mM), one of the drugs
at different concentrations, and H O (100 μM). The reaction
2
2
General Procedure for the Synthesis of Compounds 33 and 40.
To a solution of nitrile (11 mmol) in t-BuOH (40 mL) was added
finely powdered KOH (5 g, 89 mmol). The solvent was refluxed
for 2 h with stirring. Then a saturated solution of NaCl (50 mL)
was added and the resulting mixture was extracted three times
2
2
was stopped after 5 min by cooling the tubes in ice. The assay
was performed as described by Moguilevsky et al. in a NUNC
maxisorp plate (VWR, Zaventem, Belgium): 200 ng/well of
LDL was coated overnight at 4 ꢀC in a sodium bicarbonate
with CH
EtOAc 4:1 then CH Cl /methanol 1:1). The fractions obtained
2 2 2 2
Cl . Flash chromatography was performed (CH Cl /
2
2
with the second solvent mixture were evaporated.
General Procedure for the Synthesis of Compounds 34 and 41.
The amide compound (7.7 mmol) was dissolved in dioxane (50 mL)
5
8
pH 9.8 buffer (100 μL). Afterward, the plate was washed with
TBS 80 buffer and then saturated during 1 h at 37 ꢀC with the
PBS buffer containing 1% BSA (150 μL/well). After washing the
wells twice with the TBS 80 buffer, the monoclonal antibody
Mab AG9 (200 ng/well) obtained according to a standard
protocol and as previously described was added as a diluted
solution in PBS buffer with 0.5% BSA and 0.1% of Polysorbate 20.
After incubation for 1 h at 37 ꢀC, the plate was washed four
times with the TBS 80 buffer and a 3000 times diluted solution of
IgG antimouse alkaline phosphatase (Promega, Leiden, The
Netherlands) in the same buffer was added (100 μL/well). The
wells were washed again four times and a revelation solution
(150 μL/well) containing 5 mg of para-nitrophenyl phosphate in
5 mL of diethanolamine buffer was added for 30 min at room
temperature. The reaction was stopped with 60 μL/well of 3 N
NaOH solution. The measurement of the absorbance was
performed at 405 nm with a background correction at 655 nm
with a Bio-Rad photometer for a 96-well plate (Bio-Rad labora-
4
and LiAlH (1.0 M solution in dioxane, 25 mL) was added. The
suspension was refluxed for 3 h, the reaction was quenched
with ice and a 15 wt % KOH aqueous solution (10 mL). The
mixture was filtered through Celite and extracted with EtOAc
(30 mL). The organic layer was extracted with 0.1 M HCl and,
after decantation, the aqueous phase was washed with diethy-
lether. A 1 M KOH solution was added to the acidic layer (pH ≈
1
0) and this was extracted with diethylether. The solvent was
2 4
dried on Na SO and evaporated. The residue was dissolved in
diethylether, and a saturated solution of anhydrous oxalic acid
in diethylether was added dropwise. The precipitate was filtered,
washed with ether, and dried at 40 ꢀC under reduced pressure.
General Procedure for the Synthesis of Compounds 36 and 44.
A 1 M DIBAL-H solution in CH Cl (7.8 mL, 7.38 mmol) was
2
2
added slowly to a solution of the lactone (6.6 mmol) in dry
CH Cl
at -78 ꢀC. After stirring at the same temperature for 1 h,
2
2
3
0
methanol (8.9 mL) was added dropwise and the mixture poured
into 0.5 M HCl (200 mL). After stirring for additional 1 h at
room temperature, the organic layer was separated. The aque-
tories, CA, USA). Results were expressed as IC50.
Transient State Kinetics. Material. Highly purified myelo-
peroxidase of a purity index (A430/A280) of a least 0.86 was
purchased from Planta Natural Products (http://www.planta.
ous layer was extracted with CH
combined organic layers were dried with Na SO and evapo-
2
Cl
2
(3 Â 20 mL), and the
-
1
at). Its concentration was calculated using ε430 = 91 mM
cm . Hydrogen peroxide, obtained from a 30% solution was
2 4
-
1
rated. The residue purified by flash chromatography (first CH Cl ,
2
2
2 2
then CH Cl :EtOAc (60:40)). The second fraction was evaporated
diluted and the concentration determined by absorbance mea-
surement at 240 nm where the extinction coefficient is 39.4 M
-
1
to give the desired product.
Pharmacological Tests. Myeloperoxidase Inhibition Assay.
The assay was based on the production of taurine chloramine
-
1
cm . Tryptamine derivative stock solutions were prepared in
dimethylsulfoxide (DMSO) and stored in dark flasks. Dilution
was performed with 200 mM phosphate buffer, pH 7.4, to a final
-
produced by the MPO/H
2
O
selected inhibitor at defined concentration. The reaction mix-
2
/Cl system in the presence of a
4
6
v
DMF concentration of 2% ( /
v
) in all assays.
ture contained the following reagents in a final volume of 200 μL:
Transient-State Kinetics. The multimixing stopped-flow mea-
surements were performed with the Applied Photophysics (UK)
instrument SX-18MV. When 100 μL were shot into a flow cell
having a 1 cm light path, the fastest time for mixing two solu-
tions and recording the first data point was 1.3 ms. Kinetics were
followed both at single wavelength and by using 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 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.
3
-
pH 7.4 phosphate buffer (10 mM PO
(
4
/300 mM NaCl), taurine
15 mM), the compound to be tested (up to 20 μM), and the fixed
amount of the recombinant MPO (6.6 μL of MPO batch solu-
tion diluted 2.5 times, 40 nM). When necessary, the volume was
adjusted with water. This mixture was incubated at 37 ꢀC and
the reaction initiated with 10.0 μL of H
2
O
2
(100 μM). After
5
min, the reaction was stopped by the addition of 10 μL of
catalase (8 U/μL). To determine the amount of taurine chlor-
amines produced, 50 μL of 1.35 mM solution of thionitroben-
zoic acid were added and the volume adjusted to 300 μL with
water. Then, the absorbance of the solutions was measured at
4
12 nm with a microplate reader and the curve of the absorbance
as a function of the inhibitor concentration was plotted. IC50
values were then determined using standard procedures taking
into account the absence of hydrogen peroxide as 100% of
Compound I and II Formation. Conditions of MPO Com-
5
9
pound I formation were described recently. Typically, 8 μM
MPO were premixed with 80 μM H O , and after a delay time of
1
6
inhibition and the absence of inhibitors as 0% of inhibition.
2
2
2
0 ms, Compound I was allowed to react with varying concen-
Determination of LDL Oxidation Inhibition. Preparation of
the Recombinant Enzyme and of LDL. Recombinant MPO was
prepared as previously described. Each batch solution is char-
acterized by its protein concentration (mg/mL), its activity
trations of tryptamine 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 Compound II
and its subsequent reduction to native MPO by the tryptamine
derivatives (decrease in absorbance at 456 nm).
(U/mL), and its specific activity (U/mg). The chlorination
activity was determined according to Hewson and Hager.
Human plasma served for the isolation of LDL by ultracentri-
5
6
5
7
fugation according to Havel et al. Before oxidation, the LDL
fraction (1.019 < d < 1.067 g/mL) was desalted by two con-
secutive passages through PD10 gel-filtration columns (Amersham
Biosciences, The Netherlands) using PBS buffer. The different
steps were carried out in the dark, and the protein concentration
was measured by the Lowry assay for both MPO and LDL.
Acknowledgment. This study was supported by grants
from the Belgian Fund for Scientific Research (FRS-FNRS),
no. 34553.08, a grant from the FER 2007 (ULB) and a grant
from the Department of International Relationship (BRIC

8
758 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
007). We thank Prof. M. Luhmer and Dr. R. D’Orazio from
CIREM (ULB) for NMR spectra recording.
Soubhye et al.
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Supporting Information Available: synthetic procedures not
described in the Experimental Section, spectral analyses of the
compounds, purity data of the final compounds, table of inhi-
bition of the oxidation of LDL at three concentrations (1000,
100, and 50 nM). This material is available free of charge via the
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