Optimization of 5-hydroxytryptamines as dual function inhibitors targeting phospholipase A2 and leukotriene A4 hydrolase

Article abstract of DOI:10.1016/j.ejmech.2012.10.057

Dual function inhibitors targeting phospholipase A₂ (PLA ₂) and leukotriene A₄ hydrolase (LTA₄H) may balance the arachidonic acid (AA) metabolic network and be used as new anti-inflammatory drugs. In previous study, we discovered multi-target drugs towards the AA metabolic network, among which a dual-target inhibitor (JMC08-4) for human nonpancreatic secretory phospholipase A₂ (hnps-PLA ₂) and human leukotriene A₄ hydrolase (LTA₄H-h) was found. Based on the structure of compound JMC08-4, new dual-target inhibitors were designed assisted by molecular docking. In this report, a series of 5-hydroxytryptamine compounds were synthesized; and most of these title compounds showed more potent inhibitory activity than compound JMC08-4 in the in vitro bioassay against these two enzymes. The best one inhibited hnps-PLA ₂ and LTA₄H-h with IC₅₀ values of 9.2 ± 0.5 μM and 2.4 ± 1.4 μM, respectively.

Full text of DOI:10.1016/j.ejmech.2012.10.057

European Journal of Medicinal Chemistry 59 (2013) 160e167
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Optimization of 5-hydroxytryptamines as dual function inhibitors targeting
phospholipase A2 and leukotriene A4 hydrolase
*
Hu Meng, Ying Liu , Yujing Zhai, Luhua Lai
BNLMS, Center for Quantitative Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Dual function inhibitors targeting phospholipase A2 (PLA2) and leukotriene A4 hydrolase (LTA4H) may
balance the arachidonic acid (AA) metabolic network and be used as new anti-inflammatory drugs. In
previous study, we discovered multi-target drugs towards the AA metabolic network, among which
a dual-target inhibitor (JMC08-4) for human nonpancreatic secretory phospholipase A2 (hnps-PLA2) and
human leukotriene A4 hydrolase (LTA4H-h) was found. Based on the structure of compound JMC08-4,
new dual-target inhibitors were designed assisted by molecular docking. In this report, a series of
5-hydroxytryptamine compounds were synthesized; and most of these title compounds showed more
potent inhibitory activity than compound JMC08-4 in the in vitro bioassay against these two enzymes.
Received 27 June 2012
Received in revised form
26 October 2012
Accepted 27 October 2012
Available online 17 November 2012
Keywords:
5-Hydroxytryptamine
Optimization
PLA2
The best one inhibited hnps-PLA2 and LTA4H-h with IC50 values of 9.2 ꢀ 0.5
m
M and 2.4 ꢀ 1.4
mM,
respectively.
Ó 2012 Elsevier Masson SAS. All rights reserved.
LTA4H
Dual function inhibitor
1. Introduction
prostaglandins (PGs) on the other hand. Multi-target drug
discovery has become an increasingly perspective to overcome
disadvantages of single-target based drugs [5,6]. In anti-
inflammatory drug research, some multi-target inhibitors have
been reported, including combinations of COX/5-LOX [7e10], 5-
LOX/thromboxane A2 synthase (TxA2S) [11], thromboxane A2
receptor (TxA2R)/TxA2S [12], 5-LOX/microsomal prostaglandin E
synthase (mPGEs) [13], and COX/LTA4H [14].
Although multi-target drugs have advantages, there have been
no interchangeable strategies to design this new kind of drugs and
the discovery of it was often serendipitous [15]. In a previous study,
we developed a computational algorithm for finding multiple
target optimal intervention (MTOI) solutions in a disease network.
We applied MTOI to the AA network and a number of optimal
combinations of target intervention (MTOI solutions) were found,
such as: COX-2/LTA4H, COX-2/5-LOX, PGES/LTA4H, PLA2/LTA4H,
PLA2/COX-2/LTA4H, and PLA2/5-LOX/COX-2, which are both effec-
tive in controlling inflammation mediators and safe with minimal
side effects [16]. We also developed a strategy that combined
receptor-based common pharmacophores with molecular docking,
which was successfully applied to screen for dual-target inhibitors
against hnps-PLA2 and LTA4H-h [17]. Compound 1,2-amino-3-[5-
(benzyloxy)-1H-indol-3-yl] propanoic acid (JMC08-4) (Fig. 1),
whose binding conformations in proteins accommodate the
common pharmacophores, is used as lead compound against the
two enzymes. We explored structural optimizations of the JMC08-
Phospholipase A2 (PLA2) and leukotriene A4 hydrolase (LTA4H)
enzymes are important anti-inflammatory targets. PLA2 hydrolyzes
the acylester bond at the sn-2 position of phosphoglycerides and
liberates free fatty acids (predominantly arachidonic acid) and
lysophospholipids. Over expression of PLA2 plays an important role
in the inflammatory process [1,2]. The proinflammatory substance
leukotriene B4 (LTB4) derived from arachidonic acid (AA) via 5-
lipoxygenase (5-LOX) pathway. LTA4H catalyzes the rate-limiting
step in the biosynthesis of LTB4, and therefore is a potent inducer
of inflammation [3]. PLA2, LTA4H, and other key inflammation-
related proteins in the AA metabolic network, such as cyclo-
oxygenase 2 (COX-2) and 5-LOX, have been studied as targets for
anti-inflammatory drugs. Many inhibitors of these targets have
been used in treating acute and chronic inflammation. However,
inhibiting only one target of the metabolic pathways can possibly
shift the metabolism of AA towards other pathways, which may
cause side effects. For example, selective COX-2 inhibitors were
reported to have cardiovascular side effects in clinical trials [4]. The
reason is that the inhibitors increased the production of leukotri-
enes (LTs) on one hand and decreased the generation of
* Corresponding author. Fax: þ86 10 62751725.
E-mail address: liuying@pku.edu.cn (Y. Liu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.057

H. Meng et al. / European Journal of Medicinal Chemistry 59 (2013) 160e167
161
2. Results and discussion
2.1. Design of 5-HT analogs
Compound JMC08-4 was docked into the substrate binding site
of LTA4H-h and hnps-PLA2 using Autodock 4.0 (Fig. 2). In Fig. 2A, we
found that there was hydrophobic space in the L-shaped pocket of
LTA4H-h when compound JMC08-4 bound there. It suggested that
increasing the length of compound JMC08-4’s hydrophobic chain
could enhance the interaction between this compound and LTA4H-
h. Fig. 2B is the binding pocket of hnps-PLA2. We can see that using
a larger aromatic group to replace the 5-benzyloxy group of
compound JMC08-4 (e.g. compound in Fig. 3B), the binding of the
ligand to hnps-PLA2 was almost unchanged.
Molecular docking results of compound JMC08-4 gave clues on
the further optimization of LTA4H/PLA2 dual function inhibitors.
Using compound JMC08-4 backbone, a series of 5-HT analogs were
designed as LTA4H/PLA2 inhibitors. First, 5-hydroyl group of 5-HT
was combined with different aromatic groups to enhance the
hydrophobicity. One of them is 4f, which was docked into both
enzymes as shown in Fig. 3. The larger aromatic group of 4f inter-
acted with the residues Leu369, Leu367 and Pro382 of the pocket,
the interaction increased hydrophobicity as we prediction. To
further enhance the activity of these compounds, the 3-ethylamine
Fig. 1. Structure of JMC08-4 and 5-HT.
4, in relation to the binding pocket requirements of both enzymes,
5-hydroxytryptamine (serotonin, 5-HT, Fig. 1) analogs were then
selected for studying.
The 5-HT analogs are less difficulty in synthesis and possess
potential druggability. The compound 5-HT is a neurotransmitter
that modulates neural activity and a wide range of neuro-
psychological processes [18]. Some of 5-HT analogs have been
reported as agonists or antagonists of serotonin receptors. Per-
outka et al. reported that 5-benzyloxytryptamine (4a in this
article) is a known ligand of 5-hydroxytryptamine receptors [19],
and Buzzi et al. found it can mediate blockage of neurogenic
plasma extravasation in rat duramater [20]. We hope to develop 5-
HT analogs as new anti-inflammatory drugs. A series of 5-HT
analogs have been designed and tested for their inhibition
against LTA4H-h and hnps-PLA2.
Fig. 2. The binding models of compound JMC08-4. A, compound JMC08-4 with LTA4H; B, compound JMC08-4 with sPLA2.
Fig. 3. The binding models of a molecule with a longer hydrophobic group (4f) (predicted by molecular docking). A, compound 4f with LTA4H; B, compound 4f with sPLA2.

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H. Meng et al. / European Journal of Medicinal Chemistry 59 (2013) 160e167
presence of Cs2CO3, the intermediate N-BOC-5-HT (2) and different
electrophiles RnX (X is a halogen) reacted in DMF to get interme-
diates 3aeh. The title compounds 4aeh were obtained by
removing the BOC anhydride in the ethyl acetate solution of
hydrochloride. These monohydrochloride compounds were con-
verted to the title compounds 5def through nucleophilic conjugate
addition by treating with methacrylate in methanol. Another kind
of title compounds 6def were obtained from 5def by hydrolysis in
an acetone solution of NaOH.
Fig. 4. Design of new inhibitors. 1, modification with large hydrophobic groups; 2,
modification with functional groups that coordinate with metal atoms.
In the synthesis of compounds 3aeh, some electrophilic
reagents (Rf-hX) were not commercially available. Scheme 2 shows
the preparations of these complicated electrophiles. In the pres-
ence of KOH, 4-methoxyphenol and chloronitrobenzene was melt
together and condensed to get compounds Rg1 and Rh1. Stirred in
dichloromethane solution of BBr3, compounds Rg1 and Rh1were
converted to Rg2 and Rh2. When Rg2, Rh2 and phenoxyphenol were
further refluxed in 1,2-dibromoethane of KOH, corresponding
intermediates RfX, and Rg-hX were got.
group of 5-subtituied-tryptamine was combined with either func-
tional groups possessing negative charge or atoms coordinating
with metal atoms (e.g. carboxyl).
For designed potential 5-HT analogs, the two major ways of
optimization are: 1) modification with large hydrophobic groups;
2) addition of functional groups that coordinate with metal atoms
(Fig. 4).
2.2. Synthesis of 5-HT analogs
2.3. Evaluation of inhibitory activities
The fourteen 5-HT analogs (4aeh, 5def, and 6def) were
prepared by multi-step organic synthesis (Scheme 1). Primary
amino group of 5-HT was first protected by BOC [21]. Then, in the
All the title compounds were subjected to in vitro inhibitory
activities against LTA4H-h and hnps-PLA2 using the following
Scheme 1. Synthesis of compounds 4aeh, 5def, and 6def. (a), BOC anhydride, NaHCO3, NaCl, DCM; (b), RnX, Cs2CO3, DMF; (c), HCl, EtOAc, rt; (d), methyl acrylate, Na2CO3, MeOH;
(e), NaOH, acetone, water.
Scheme 2. Preparation of complicated electrophiles Rf-hX. (a), KOH; (b), BBr3, CH2Cl2; (c), 1,2-dibromoethane, KOH.

H. Meng et al. / European Journal of Medicinal Chemistry 59 (2013) 160e167
163
Table 1
Inhibitory activity of compounds 4aeh, 5def, and 6def.
R1
O
HN R2
N
H
Compound
R1
R2
LTA4H IC50
(
m
M)
PLA2 IC50 (mM)
Aminopeptidase
Epoxide hydrolase
Bestatin
NI101
JMC08-4
4a
e
e
e
e
e
e
H
4.2 ꢀ 0.5
>100
>100
>100
0.88 ꢀ 0.15
42.1 ꢀ 1.9
52 ꢀ 5
17.3 ꢀ 0.2
231 ꢀ 4
13 ꢀ 1
>100
4b
4c
H
H
8.7 ꢀ 0.8
30 ꢀ 5
>100
>100
25 ꢀ 5
27 ꢀ 9
4d
H
2.1 ꢀ 0.2
18 ꢀ 4
4.4 ꢀ 0.9
4e
4f
H
H
8.9 ꢀ 0.5
1.3 ꢀ 0.2
>100
15.3 ꢀ 0.4
11.5 ꢀ 0.9
O
O
15 ꢀ 3
O
O
O
4g
4h
H
H
41.8 ꢀ 0.9
6.8 ꢀ 0.6
>100
>100
5.3 ꢀ 0.6
8.6 ꢀ 0.9
O2N
O
NO2
O
5d
COOMe
5.8 ꢀ 0.5
7.6 ꢀ 0.5
38 ꢀ 5
5e
5f
COOMe
COOMe
9.27 ꢀ 0.09
0.67 ꢀ 0.03
15.8 ꢀ 5.3
2.4 ꢀ 1.4
25 ꢀ 6
O
O
9.2 ꢀ 0.5
O
6d
COOH
7.08 ꢀ 0.01
3.8 ꢀ 0.7
53 ꢀ 12
6e
6f
COOH
COOH
9.9 ꢀ 0.7
5.2 ꢀ 0.1
>100
>100
58 ꢀ 9
O
O
71 ꢀ 12
O
methods. The inhibitory activity results are summarized in Table 1.
Limited by solubility, only compounds with 50% inhibition below
100 mM were tested for IC50 values.
could be used to evaluate inhibitors’ binding affinities. Using previ-
ously reported methods [23,24], we measured LTA4H inhibitory
activities of these compounds against both aminopeptidase and
epoxide hydrolase, with bestatin as the positive control.
2.3.1. Against LTA4H
LTA4H is a ubiquitously distributed 69 kDa zinc-containing cyto-
solic enzyme. It possesses both aminopeptidase activity and epoxide
hydrolaseactivity[22], andthesubstratebindingpocketsofthesetwo
activities are largely overlap. The activity assay of LTA4H aminopep-
tidase is more sensitive to inhibitor binding and less expensive. When
the weak inhibitors showed no reliable response in the epoxide
hydrolase activity assay, the activity data of LTA4H aminopeptidase
2.3.2. Against PLA2
Testing inhibitory activity of the compounds on hnps-PLA2 was
based on a continuous fluorescence assay and carried out in a 96-
well plate, using 1-anilinonaphthalene-8-sulfonate (ANS) as an
interfacial probe [25]. 2-(1-Benzyl-5-methoxy-2-methyl-1H-indol-
3-yl) acetamide (NI101), one reported hnps-PLA2 inhibitor [26],
was used as the positive control.

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H. Meng et al. / European Journal of Medicinal Chemistry 59 (2013) 160e167
2.4. Structure activity relationships
aluminum sheets and UV light (254 and 366 nm) for detection. All
title compounds gave satisfactory 1H NMR, 13C NMR, elemental
analyses, and mass spectrometry analyses. The 1H NMR and 13C
NMR spectra were measured on a Bruker-400 M spectrometer
using TMS as internal standard. Elemental analyses were per-
formed on an ElementarVario EL instrument. Mass spectra were
recorded on a VG-ZAB-HS spectrometer. Title compounds 4aef
possessed a purity of >99.5% as verified by elemental analyses after
comparison with calculated values.
As shown in Table 1, most compounds displayed more potent
inhibitory activity against LTA4H-h and hnps-PLA2, than compound
JMC08-4 did.
In the series of secondary amine compounds (compounds 4aef),
compounds 4d and 4f show more potent inhibitory activity than
the other compounds against LTA4H-h and hnps-PLA2, with IC50
values in the magnitude of 10
mM. These results suggested that
compounds with longer hydrophobic groups would show higher
activities than those with smaller ones, which were consistent with
the molecular docking results. However, compounds 4g and 4h
with nitro-substituted aromatic groups did not show good inhibi-
tion against LTA4H because these groups were not fit well into the
L-shaped narrow pocket.
4.1.1. General procedure for the synthesis of N-(butyloxycarbonyl)
serotonin (2)
Serotonin hydrochloride (1.07 g, 5 mmol) was suspended in
10 mL of chloroform. Sodium bicarbonate (0.42 g, 10 mmol) in 7 mL
of water, 1 g of sodium chloride, and di-tert-butyloxycarbonyl
anhydride (1.1 g, 5 mmol) in 2.5 mL of chloroform were added.
The mixture was refluxed for 3 h. The reaction mass was filtered
and aqueous phase was washed with chloroform. The combined
organic phases were washed with water and brine and dried over
magnesium sulfate. The solvent was removed under reduced
pressure to produce a solid product. Yield: 90%. 1H NMR (CDCl3):
Compounds 5def showed stronger LTA4H inhibitory activities
than compounds 4aeh due to their functional groups coordinating
with metal atoms. The interaction between the zinc ion and oxygen
atoms of the methyl propionate group may cause the enhancement
of their binding affinities. In contrast, compounds 6def, with the
propionic acid group at position 2 showed little improvement in
inhibitory activity against LTA4H. Propionic acid group possessed
both negative charge and atoms that could coordinate with metal
atoms and was supposed to improve the binding affinity of
compounds 6def. It may have been caused by the intramolecular
hydrogen bonds between the carboxyl group and the amino group.
Except for compound 5f, all compounds with a substitution at
position 2 had decreased inhibitory activity against PLA2 about one
order of magnitude. It is probably caused by unwanted collisions
appeared between the inhibitors and some residues of PLA2.
Though multi-target inhibitors generally do not have high affini-
ties, they have potential beneficial effects, such as avoiding drug
resistance and toxicity, and stabilizing “sick” cells [27]. For example,
d
7.97 [s (br), 1H], 7.26 (d, 1H), 7.01 (d þ s, 2H), 6.80 (dd, 1H), 4.66 (s
(br), 1H), 3.42 (t þ m, 2H), 2.87 (t, 2H), 1.44 (s, 9H).
4.1.2. General procedure for the preparation of 1-(4-
methoxyphenoxy)-2-nitrobenzene (Rh1
)
A mixture of 4-methoxyphenol (2.0 g, 16 mmol) and anhydrous
potassium hydroxide (1.0 g, 18 mmol) was heated at 150 ꢁC and
stirred. After the mixture melted (about 10 min), 1-chloro-2-
nitrobenzene (2.0 g, 13 mmol) was added and the mixture was
stirred at 170 ꢁC for 2 h, while being monitored by TLC. When the
reaction was complete, the reaction mass was poured into 50 mL of
3% potassium hydroxide aqueous solution and stirred at room
temperature for about 2 h and then cooled to 4 ꢁC. The solid was
filtered with suction and washed with water. The crude product
was recrystallized by ethanol to afford yellow solid 2.8 g (yield
noncompetitive antagonists of N-methyl-D-aspartate receptors only
inhibit multiple targets weakly, but exhibit advantages of reducing
side effects [28e31]. We would therefore expect that these multi-
target inhibitors may be more effective to control inflammation
than single-target inhibitors. Of course, these compounds need to be
further studied to characterize their overall effects at system level.-
90%). 1H NMR (400 MHz, DMSO-d6):
d 7.93 (1H, d), 7.44 (1H, m),
7.26e6.90 (6H, m), 3.82 (3H, s).
Rg1 was synthesized by a similar procedure, which was moni-
tored by TLC.
3. Conclusion
4.1.3. General procedure for the preparation of 4-(2-nitrophenoxy)
In this report, we aimed to identify potent dual function inhibi-
tors by improving the binding affinities of a known moderate
inhibitor of PLA2 and LTA4H, compound JMC08-4. Following docking
result and structural information of this compound, two major
optimizations were made: 1) hydrophobic group at position 5 of
indole backbone was enlarged to increase the hydrophobic interac-
tion; and 2) functional group that could coordinate with metal atoms
was added at proper site. These optimizations turned out to be
effective. Fourteen 5-HT analogs were successfully designed and
synthesized. Most of these compounds showed more potent inhib-
itory activity against LTA4H-h and hnps-PLA2 than compound
JMC08-4. The best potential compound is 5f, which inhibited LTA4H-
phenol (Rh2)
Rh1 (1.0 g, 4 mmol) was stirred in CH2Cl2 (20 mL) at 0 ꢁC, and
0.57 mL (6 mmol) BBr3 in 20 mL CH2Cl2 was slowly dropped into
the solution above. After being stirred at room temperature for
approximately 3 h, the mixture was poured into H2O. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
at reduced pressure. The residue was separated by flash chroma-
tography (silica gel, the eluting solvent ranged from hexane to
EtOAc/hexane ¼ 1:2) as a yellow solid which weighed 0.87 g (yield
92%). 1H NMR (400 MHz, DMSO-d6):
d 9.50 (1H, s), 8.00 (1H, d), 7.60
(1H, m), 7.24 (1H, m), 6.97e6.80 (5H, m).
Rg2 was synthesized by a similar procedure, which was moni-
tored by TLC.
h with an IC50 value of 2.4 ꢀ 1.4
mM, increasing nearly three orders of
magnitude than compound JMC08-4. It also improved inhibitory
activity against hnps-PLA2, the IC50 value is reach to 9.2 ꢀ 0.5
mM.
4.1.4. General procedure for the preparation of 1-(4-(2-
bromoethoxy) phenoxy) benzene (RfX)
4. Experimental
The compound 4-phenoxyphenol (250 mg, 1.34 mmol) and
anhydrous potassium hydroxide (150 mg, 2.66 mmol) were
refluxed in 1,2-dibromoethane (2 mL) for 8 h. The mixture was
washed with water and brine, and then dried over magnesium
sulfate. After being concentrated at reduced pressure, the residue
was separated by flash chromatography (silica gel, the eluting
solvent ranged from hexane to EtOAc/hexane ¼ 3:7) as a colorless
4.1. Chemistry
The reagents and solvents were commercially available and
purified according to conventional methods. All reactions were
monitored by thin layer chromatography; using silica gel 60 F-254

H. Meng et al. / European Journal of Medicinal Chemistry 59 (2013) 160e167
165
liquid and weighed 313 mg (yield 80%). 1H NMR (400 MHz, CDCl3):
(CN), 131.63 (Cindole), 132.46, 132.80, 135.38 (CN), 152.09 (OeCindole);
ESI-HRMS m/z 317.16435. Anal. Calcd for C21H21ClN2O: C, 71.48; H,
6.00; N, 7.94. Found: C, 71.01; H, 6.00; N, 7.83.
d
7.31 (2H, t þ s), 7.06 (1H, t), 6.98 (6H, m), 4.28 (2H, t), 3.65 (2H, t).
RgX and RhX were synthesized by a similar procedure, which
was monitored by TLC.
4.1.7.3. 2-(5-(Naphthalen-1-ylmethoxy)-1H-indol-3-yl) ethanami-
4.1.5. General procedure for the preparation of tert-butyl 2-(5-(2-
(4-phenoxyphenoxy) ethoxy)-1H-indol-3-yl) ethylcarbamate (3f)
A mixture of 2 (126 mg, 0.457 mmol) and RfX (147 mg,
0.502 mmol) was stirred in anhydrous DMF (4 mL) in the presence
of Cs2CO3 (140 mg, 0.430 mmol) at 90 ꢁC for 8 h. The mixture was
poured into ethyl acetate (20 mL) and washed with water and
brine, then dried over magnesium sulfate. After being concentrated
at reduced pressure, the residue was separated by flash chroma-
tography (silica gel, the eluting solvent ranged from EtOAc/
hexane ¼ 1:9 to EtOAc/hexane ¼ 2:3) as a colorless oil which
nium chloride (4c). White solid, yield 89%, 1H NMR (400 MHz,
DMSO-d6):
1H), 7.55 (m, 3H), 7.36 (s, 1H), 7.28 (d, 1H), 7.21 (d, 1H), 6.86 (m, 1H),
5.29 (s, 2H), 3.01 (m, 4H); 13C NMR (DMSO-d6):
23.12 (CH2), 69.50
d 10.85 (s, 1H), 8.15 (d, 1), 7.92e8.02 (m, 6H), 7.72 (d,
d
(OeCH2), 101.84, 109.29, 111.85, 112.17, 124.09 (Cindole), 125.36,
125.88, 126.17, 126.59 (CN), 127.21 (Cindole), 127.56, 127.75, 127.90,
128.40, 131.23 (CN), 131.65 (Cindole), 133.20 (CN), 152.08 (OeCindole);
ESI-HRMS m/z 317.16454. Anal. Calcd for C21H21ClN2O: C, 71.48; H,
6.00; N, 7.94. Found: C, 71.00; H, 6.02; N, 7.75.
weighed 120 mg (yield 59%). 1H NMR (400 MHz, CDCl3):
d
7.94 (s
4.1.7.4. 2-(5-(4-Phenylbenzyloxy)-1H-indol-3-yl)
chloride (4d). White solid, yield 91%, 1H NMR (400 MHz, DMSO-d6).
10.88 (s, 1H), 8.16 (s (br), 3H), 7.68 (dd, 4H), 7.59 (d, 2H), 7.47 (t,
2H), 7.37 (t, 1H), 7.28 (d, 1H), 7.21 (d, 2H), 6.84 (dd, 1H), 5.17 (s, 2H),
2.98 (m, 4H); 13C NMR (DMSO-d6):
23.11 (CH2), 69.45 (OeCH2),
ethanaminium
(br), 1H), 7.26 (m, 2H), 7.10 (s, 1H), 7.04 (m, 2H), 6.97 (m, 7H), 4.61 (s
(br), 1H), 4.35 (dd, 4H), 3.45 (s (br), 2H), 2.91 (t, 2H), 1.43 (s, 9H).
The compounds 3a, 3c, and 3eeh were synthesized by a similar
procedure, which was monitored by TLC.
d
d
101.76, 109.29, 111.81, 112.16, 124.06 (Cindole), 126.63 (4C, Cph),
127.17 (Cindole), 127.56 (Cph), 128.31, 128.91 (2C, Cph), 131.59 (Cindole),
136.97, 139.47, 139.85(Cph), 152.05 (OeCindole); ESI-HRMS m/z
343.18014. Anal. Calcd for C23H23ClN2O: C, 72.91; H, 6.12; N, 7.39.
Found: C, 72.89; H, 6.10; N, 7.27.
4.1.6. General procedure for the preparation of tert-butyl 2-(5-
(naphthalen-2-ylmethoxy)-1H-indol-3-yl) ethylcarbamate (3b)
A mixture of compound 2 (292 mg, 1.06 mmol) and 2-(bromo-
methyl) naphthalene (141 mg, 1.00 mmol) was stirred in anhydrous
DMF (4 mL) in the presence of Cs2CO3 (420 mg, 1.5 mmol) at room
temperature for 8 h. The mixture was poured into ethyl acetate
(20 mL) and washed with water and brine, then dried over
magnesium sulfate. After being concentrated at reduced pressure,
the residue was separated by flash chromatography (silica gel, the
4.1.7.5. 2-(5-(3-Phenoxybenzyloxy)-1H-indol-3-yl)
chloride (4e). White solid, yield 88%, 1H NMR (400 MHz, DMSO-
d6): 10.87 (s, 1H), 8.11 (s (br), 3H), 7. 43 (m, 3H), 7.18 (m, 6H), 6.98
(dd, 3H), 6.80 (d, 1H), 5.11 (s, 2H), 2.96 (dd, 4H); 13C NMR (DMSO-
d6): 23.07 (CH2), 69.42 (OeCH2), 101.95, 109.26, 111.77, 112.12
ethanaminium
d
eluting solvent ranged from EtOAc/hexane
hexane ¼ 2:3) as a colorless oil which weighed 290 mg (yield 66%).
1H NMR (400 MHz, CDCl3):
8.11 (s (br), 1H), 7.92 (s, 1H), 7.84 (m,
¼
1:9 to EtOAc/
d
(Cindole), 117.40, 117.61 (Cph), 118.69 (2C, Cph), 122.47, 123.51 (Cph),
124.10, 127.17 (Cindole), 129.99 (Cph), 130.04 (2C, Cph), 131.66 (Cindole),
140.10 (Cph), 151.92 (OeCindole), 156.47, 156.73(OeCph); ESI-HRMS
m/z 359.17468. Anal. Calcd for C23H23ClN2O2: C, 69.95; H, 5.87; N,
7.09. Found: C, 69.62; H, 5.86; N, 7.09.
d
3H), 7.58 (d, 1H), 7.47 (m, 2H), 7.22 (d, 1H), 7.18 (s, 1H), 6.97 (m, 2H),
5.25 (s, 2H), 4.65 (s (br), 1H), 3.43 (d, 2H), 2.89 (t, 2H), 1.44 (s, 9H).
The compound 3d was synthesized by a similar procedure,
which was monitored by TLC.
4.1.7.6. 2-(5-(2-(4-Phenoxyphenoxy) ethoxy)-1H-indol-3-yl) etha-
4.1.7. General procedure for the preparation of 2-(5-(benzyloxy)-
1H-indol-3-yl) ethanaminium chloride (4a)
naminium chloride (4f). White solid, yield 90%, 1H NMR (400 MHz,
DMSO-d6):
7.20 (dd, 2H), 7.04 (m, 5H), 6.93 (d, 2H), 6.79 (dd, 1H), 4.00 (s, 4H),
3.03 (m, 4H); 13C NMR (DMSO-d6):
23.07 (CH2), 66.96, 79.00 (Oe
d 10.88 (s, 1H), 8.14 (s, 3H), 7.32 (m, 3H), 7.32 (m, 3H),
The compound 3a (183 mg, 0.5 mmol) was dissolved in anhy-
drous ethyl acetate saturated with hydrogen chloride (6 mL). A
white precipitate appeared while the solution was stirred and
bubbled with dry HCl gas for 45 min. The solid was filtered with
suction and washed with ethyl acetate, and the crude product was
recrystallized by water as a white powder which weighed 166 mg
(yield 90%).
d
CH2), 101.37, 109.32, 111.65, 112.20 (Cindole), 115.78, 117.29, 120.69
(2C, Cph), 122.59 (Cph), 124.09, 127.19 (Cindole), 129.85 (2C, Cph),
131.60 (Cindole), 149.53 (Cph), 152.05 (OeCindole), 154.77, 157.94(Oe
Cph); ESI-HRMS m/z 389.18504. Anal. Calcd for C24H25ClN2O3: C,
67.84; H, 5.93; N, 6.59. Found: C, 67.48; H, 5.94; N, 6.53.
The compounds 4beh were synthesized by a similar procedure.
4.1.7.7. 2-(5-(2-(4-(4-Nitrophenoxy) phenoxy) ethoxy)-1H-indol-3-
4.1.7.1. 2-(5-(Benzyloxy)-1H-indol-3-yl) ethanaminium chloride
yl) ethanaminium chloride (4g). Pale yellow solid, yield 63%, 1H
(4a). White solid, yield 90%, 1H NMR (400 MHz, DMSO-d6):
d
10.87
(s, 1H), 8.16 (s, 3H), 7.49 (d, 2H), 7.29 (m, 6H), 6.82 (dd, 1H), 5.11 (s,
2H), 3.01 (m, 4H); 13C NMR (DMSO-d6):
23.08 (CH2), 69.83 (Oe
NMR (400 MHz, DMSO-d6):
7.28 (d, 1H), 7.13 (m, 8H), 6.80 (d, 1H), 4.36 (s, 4H), 3.03 (m, 4H); 13
NMR (DMSO-d6): 23.07 (CH2), 66.85, 79.06 (OeCH2), 101.38,
d 10.88 (s, 1H), 8.26 (d, 2H), 8.21 (s, 3H),
C
d
d
CH2), 101.74, 109.27, 111.81, 112.14, 124.04, 127.15 (Cindole), 127.60
(Cph), 127.69, 128.31 (2C, Cph), 131.57 (Cindole), 152.08 (OeCindole);
ESI-HRMS m/z 267.14865. Anal. Calcd for C17H19ClN2O: C, 67.43;
H, 6.32; N, 9.25. Found: C, 67.09; H, 6.27; N, 9.21.
109.32, 111.64, 112.20 (Cindole), 116.11, 116.58, 121.97 (2C, Cph), 124.09
(Cindole), 126.12 (2C, Cph), 127.19, 131.61 (Cindole), 141.42, 147.42 (Cph),
152.03 (OeCindole), 154.97, 163.72(OeCph); ESI-HRMS m/z
434.17094.
4.1.7.2. 2-(5-(Naphthalen-2-ylmethoxy)-1H-indol-3-yl) ethanami-
4.1.7.8. 2-(5-(2-(4-(2-Nitrophenoxy) phenoxy) ethoxy)-1H-indol-3-
nium chloride (4b). White solid, yield 67%, 1H NMR (400 MHz,
yl) ethanaminium chloride (4h). Pale yellow solid, yield 74%, 1H
DMSO-d6):
7.63 (dd, 1H), 7.52 (m, 2H), 7.29 (dd, 2H), 7.21 (d, 1H), 6.87 (dd, 1H),
5.29 (s, 2H), 3.03 (m, 4H); 13C NMR (DMSO-d6):
23.09 (CH2), 70.00
d
10.88 (s, 1H), 8.15 (s, 3H), 8.04 (s, 1H), 7.93 (m, 3H),
NMR (400 MHz, DMSO-d6):
3H), 7.18 (m, 6H), 6.98 (dd, 3H), 6.81 (d, 1H), 5.11 (s, 4H), 3.00 (dd,
4H); 13C NMR (DMSO-d6):
23.06 (CH2), 66.87, 79.06 (OeCH2),
d 10.87 (s, 1H), 8.11 (m, 3H), 7.40 (q,
d
d
(OeCH2), 101.95, 109.28, 111.84, 112.15, 124.09 (Cindole), 125.85,
125.98, 126.16, 126.23 (CN), 127.18 (Cindole), 127.56, 127.75, 127.90
101.38, 109.32, 111.64, 112.20 (Cindole), 115.98 (2C, Cph), 119.20
(Cph), 120.71 (2C, Cph), 123.17 (Cph), 124.07 (Cindole), 125.45 (Cph),

166
H. Meng et al. / European Journal of Medicinal Chemistry 59 (2013) 160e167
127.19, 131.61 (Cindole), 134.78, 140.50, 148.49, 150.48 (Cph), 152.03
(OeCindole), 155.48 (OeCph); ESI-HRMS m/z 434.17016.
The compounds 6e and 6f were synthesized by a similar
procedure.
4.1.8. General procedure for the preparation of methyl 3-(2-(5-(4-
phenylbenzyloxy)-1H-indol-3-yl) ethylamino) propanoate (5d)
Compound 4d (378 mg, 0.679 mmol), Cs2CO3 (111 mg,
4.1.9.1. 3-(2-(5-(3-Phenoxybenzyloxy)-1H-indol-3-yl) ethylamino)
propanoic acid (6e). White solid, yield 37%, 1H NMR (400 MHz,
DMSO-d6):
7.01 (d, 2H), 6.94 (d, 1H), 6.78 (dd, 1H), 5.10 (s, 2H), 2.98 (t, 4H), 2.89
(m, 2H), 2.20 (t, 2H); 13C NMR (DMSO-d6):
23.37, 44.32, 47.18
d 10.77 (s, 1H), 7.39 (dd, 3H), 7.24 (m, 2H), 7.13 (dd, 4H),
0.575 mmol), and methyl acrylate (68 mL, 0.68 mmol) were stirred
in methanol (2 mL) at room temperature for 12 h. The mixture was
poured into ethyl acetate and washed with water and brine, then
dried over magnesium sulfate. After being concentrated at reduced
pressure, the residue was separated by flash chromatography (silica
gel, the eluting solvent ranged from CH2Cl2 to MeOH/CH2Cl2 ¼ 1:5)
as a colorless oil which weighed 226 mg (yield 78%). The
compounds 5e and 5f were synthesized by a similar procedure.
d
(CH2), 69.36 (OeC), 101.93, 110.35, 111.70, 112.03 (Cindole), 117.40,
117.60 (Cph), 118.67 (2C, Cph), 122.44, 123.49 (Cph), 123.82, 127.24
(Cindole), 129.97 (C, Cph), 130.03 (3C, Cph), 131.62 (Cindole), 140.16
(Cph), 151.80 (OeCindole), 156.48, 156.71(OeCph), 173.66 (C]O); ESI-
HRMS m/z 431.19692 [M þ H].
4.1.9.2. 3-(2-(5-(2-(4-Phenoxyphenoxy) ethoxy)-1H-indol-3-yl) eth-
ylamino) propanoic acid (6f). White solid, yield 40%, 1H NMR
4.1.8.1. Methyl 3-(2-(5-(4-phenylbenzyloxy)-1H-indol-3-yl) ethyl-
amino) propanoate (5d). Yield 78%. 1H NMR (400 MHz, CDCl3):
(400 MHz, DMSO-d6):
(s, 1H), 7.05 (d, 6H), 6.93 (d, 2H), 6.77 (d, 1H), 4.32 (s, 4H), 2.99 (t,
2H), 2.89 (t, 4H), 2.19 (t, 2H); 13C NMR (DMSO-d6):
23.44, 30.64,
d 10.75 (s, 1H), 7.35 (t, 2H), 7.25 (d, 1H), 7.17
d
8.05 (s, 1H), 7.59 (m, 6H), 7.44 (t, 2H), 7.34 (t,1H), 7.25 (m, 1H), 7.17
(d, 1H), 6.97 (m, 2), 5.15 (s, 2H), 3.62 (s, 3H), 2.93 (m, 6H), 2.50 (t,
2H); 13C NMR (CDCl3):
25.79, 34.63, 44.98, 49.76 (CH2), 51.55,
d
43.58, 44.32, 47.23 (CH2), 66.85, 66.98 (OeC), 110.43, 111.56, 112.09,
114.53 (Cindole), 115.78, 117.29, 120.69 (2C, Cph), 122.56 (Cph), 123.82,
127.29 (Cindole), 129.85 (2C, Cph), 132.60 (Cindole), 149.51 (Cph), 151.94
(OeCindole), 154.45, 157.95(OeCph), 173.03 (C]O); ESI-HRMS m/z
461.20681 [M þ H].
d
70.77 (OeC), 102.46, 111.86, 112.92, 113.73, 122.82 (Cindole), 127.13
(4C, Cph), 127.29 (Cindole), 127.87 (Cph), 128.10, 128.78 (2C, Cph),
131.78 (Cindole), 136.75, 140.75, 140.93(Cph), 153.11 (OeCindole),
173.17 (C]O); ESI-HRMS m/z 429.21640 [M þ H].
4.2. Biochemical methods
4.1.8.2. Methyl 3-(2-(5-(3-phenoxybenzyloxy)-1H-indol-3-yl) ethyl-
amino) propanoate (5e). Yield 72%. 1H NMR (400 MHz, CDCl3):
4.2.1. Inhibition assay of aminopeptidase activity of LTA4H-h
The aminopeptidase activity was determined as described
previously [17]. The enzyme was incubated at 37 ꢁC in 96-well
microtiter plates in 50 mM TriseHCl, pH 8.0, containing 100
manacles. The reactions were initiated by the addition of Ala-p-
nitroanilides as substrate. The formation of p-NA was then moni-
tored at 37 ꢁC for 15 min at 405 nm using a multi well ultraviolet
spectrometer (Spectra Max 190, Molecular Devices). For IC50
determinations, 0.125 mM substrate was used. All reactions con-
taining inhibitors were performed after incubating inhibitors with
enzymes for 2 min. The final concentration of DMSO was 5% (v/v).
The initial reaction rates at different inhibitor concentrations were
used for calculating the IC50 values.
d
8.03 (s, 1H), 7.28 (m, 5H), 7.11 (dd, 3H), 6.96 (t, 5H), 5.08 (s, 2H),
3.62 (s, 3H), 2.91 (m, 6H), 2.50 (t, 2H); 13C NMR (CDCl3):
d
25.73,
34.59, 44.96, 49.71 (CH2), 51.56, 70.61 (OeC), 102.53, 111.84, 112.91,
113.67 (Cindole), 117.94, 118.09 (Cph), 118.94 (2C, Cph), 122.28, 122.83
(Cph), 123.28, 127.83 (Cindole), 129.75 (2C, Cph), 129.83 (2C, Cph),
131.79 (Cindole), 139.79 (Cph), 152.94 (OeCindole), 156.13, 156.45 (Oe
C
ph), 173.17 (C]O); ESI-HRMS m/z 445.21206 [M þ H].
4.1.8.3. Methyl 3-(2-(5-(4-phenylbenzyloxy)-1H-indol-3-yl) ethyl-
amino) propanoate (5f). Yield 69%. 1H NMR (400 MHz, CDCl3):
d
8.03 (s, 1H), 7.27 (m, 3H), 7.12 (d, 1H), 6.97 (m, 9H), 4.35 (dq, 4H),
3.62 (s, 3H), 2.94 (m, 6H), 2.51 (t, 2H); 13C NMR (CDCl3):
d
25.74,
34.60, 44.97, 49.77 (CH2), 51.57, 67.36, 67.64 (OeC), 102.42, 111.88,
112.91, 113.70 (Cindole), 115.88, 117.68, 120.78 (2C, Cph), 122.48 (Cph),
122.86, 127.86 (Cindole), 129.62 (2C, Cph), 131.86 (Cindole), 150.46
(Cph), 152.89 (OeCindole), 155.08, 158.44 (OeCph), 173.17 (C]O); ESI-
HRMS m/z 475.22250 [M þ H].
4.2.2. Inhibition assay of hydrolase activity of LTA4H-h
The epoxide hydrolase activity was determined using an ELISA
assay to quantify the amount of LTB4 [17]. LTA4 methyl ester
(Cayman Chemical) was hydrolyzed in cold acetone with 50 mM
NaOH (20%, v/v) under an inert atmosphere of nitrogen. To deter-
mine the effect of small molecules on the epoxide hydrolase
activity of LTA4H-h, 300 ng of enzyme was incubated with
compounds in reaction buffer (10 mM sodium phosphate, pH 7.4,
4 mg/mL BSA, 5% v/v DMSO) for 15 min at 37 ꢁC The reaction was
initiated by the addition of LTA4 (150 nM final assay concentration)
4.1.9. General procedure for the preparation of 3-(2-(5-(4-
phenylbenzyloxy)-1H-indol-3-yl) ethylamino) propanoic acid (6d)
The compound 5d (226 mg, 0.527 mmol) and sodium hydroxide
(80 mg, 2 mmol) were dissolved in a mixture solution (acetone and
water, v:v ¼ 3:1) and stirred at room temperature for 12 h. The
solution was acidified to pH ¼ 1with 1 M hydrochloric acid and
extracted with ethyl acetate three times. The organic layer was
washed with brine, then dried over Na2SO4, concentrated at
reduced pressure, and the residue was separated by flash chro-
matography (silica gel, the eluting solvent ranged from CH2Cl2/
MeOH ¼ 5:1 to MeOH) as white solid which weighed 107 mg (yield
and incubated for another 10 min at 37 ꢁC. 25
to 500 L assay buffer without DMSO to stop the reaction and then
mL sample was added
m
further diluted fivefold. LTB4 was quantified in the diluted sample
by a commercially available LTB4 ELISA kit (Cayman Chemical).
4.2.3. Inhibitory activity assay of hnps-PLA2
47%). 1H NMR (400 MHz, DMSO-d6):
d
10.84 (s, 1H), 7.68 (t, 4H),
Reaction buffer (150
mL in 50 mM TriseHCl, pH 8.0, 100 mM
7.58 (d, 2H), 7.47 (t, 2H), 7.37 (t,1H), 7.28 (d, 2H), 7.20 (s,1H), 6.85 (d,
NaCl, 5 g/mL bovine serum albumin (BSA), and 10 mM ANS), 20 mL
of substrate stock solution [2 mM 1,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC)] and 2 mM of sodium deoxycholate in
m
1H), 5.17 (s, 2H), 3.15 (d, 4H), 3.04 (m, 2H), 2.68 (t, 2H); 13C NMR
(DMSO-d6):
d 21.89, 30.61, 42.66, 47.11 (CH2), 69.50 (OeC),
101.90, 109.14, 111.82, 112.15, 124.03 (Cindole), 126.62 (4C, Cph),
127.12 (Cindole), 127.42 (Cph), 128.30, 128.91 (2C, Cph), 131.60 (Cindole),
136.97, 139.48, 139.84 (Cph), 152.06 (OeCindole), 172.08 (C]O); ESI-
HRMS m/z 415.20104 [M þ H].
water, sonicated for 2 min, 10
and 10 L of inhibitor stock solution (dissolved in DMSO) were
incubated at 25 ꢁC for 10 min. Reactions were started by adding
10 L of hnps-PLA2 stock solution (3.2 g/mL) and monitored by
mL of CaCl2 stock solution (100 mM),
m
m
m

H. Meng et al. / European Journal of Medicinal Chemistry 59 (2013) 160e167
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Acknowledgments
This research was supported by the Ministry of Science
and Technology of China (grant numbers: 2009CB918503,
2012AA020308) and the National Natural Science Foundation of
China (grant numbers: 20873003, 11021463).
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