N-1, C-3 substituted indoles as 5-LOX inhibitors - In vitro enzyme immunoaasay, mass spectral and molecular docking investigations

Article abstract of DOI:10.1016/j.bmcl.2012.12.068

Based upon the structures of some known 5-LOX inhibitors, a set of five compounds carrying appropriate substituents at N-1 and C-3 of indole were synthesized and investigated for 5-LOX inhibitory activities. Fifty percent inhibitory concn (IC₅₀) of these compounds ranges from 0.6 to 5 μM and found to be comparable to that of clinically used 5-LOX inhibitor, zileuton. The compounds under present investigations exhibited appreciable interactions with 5-LOX as apparent from their association constants calculated from the mass spectral data. Compound 5a with a tosyl group at N-1 and pyrolidinyl-1,2-dione substituent at C-3 of indole, exhibiting IC₅₀ 0.6 μM and stoichiometry of 1:7 in the enzyme-compound complex was identified as highly potent 5-LOX inhibitor and seems to be suitable for further investigations.

Full text of DOI:10.1016/j.bmcl.2012.12.068

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1433–1437
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-1, C-3 substituted indoles as 5-LOX inhibitors—In vitro enzyme
immunoaasay, mass spectral and molecular docking investigations
⇑
Palwinder Singh , Pooja
Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Based upon the structures of some known 5-LOX inhibitors, a set of five compounds carrying appropriate
substituents at N-1 and C-3 of indole were synthesized and investigated for 5-LOX inhibitory activities.
Fifty percent inhibitory concn (IC50) of these compounds ranges from 0.6 to 5 lM and found to be com-
parable to that of clinically used 5-LOX inhibitor, zileuton. The compounds under present investigations
exhibited appreciable interactions with 5-LOX as apparent from their association constants calculated
from the mass spectral data. Compound 5a with a tosyl group at N-1 and pyrolidinyl-1,2-dione substitu-
Received 10 August 2012
Revised 13 December 2012
Accepted 20 December 2012
Available online 3 January 2013
Keywords:
ent at C-3 of indole, exhibiting IC50 0.6 lM and stoichiometry of 1:7 in the enzyme–compound complex
was identified as highly potent 5-LOX inhibitor and seems to be suitable for further investigations.
5-Lipoxygenase
Indole derivatives
Inhibitors
Ó 2012 Elsevier Ltd. All rights reserved.
Mass spectra
Docking
Due to the production of pro-inflammatory prostaglandins and
leukotrienes, arachidonic acid (AA) metabolism is being the target
of the drugs used for treatment of allergic and inflammatory dis-
eases.1 While prostaglandins are generated through COX-1 and
COX-2 mediated channels2 of AA metabolism, leukotrienes are pro-
duced by 5-lipoxygenase (5-LOX) through the participation of 5-
LOX activating protein (FLAP).3 Besides allergy and inflammation,
leukotrienes also play pivotal role in instigation of asthma, hyper-
sensitivity, atherosclerosis, stroke, osteoporosis as well as certain
types of cancer.4 Hence, decreasing leukotriene biosynthesis
through the inhibition of 5-LOX has been extensively studied as a
potential target for the development of novel therapies of 5-LOX
associated diseases.4a,5 Four different classes of 5-lipoxygenase
inhibitors have been identified as redox inhibitors, iron chelating
agents, active site-directed inhibitors or competitive reversible
inhibitors and inhibitors of FLAP.3c,6 To the best of our knowledge,
zileuton and a few cysteinyl-LT receptor antagonists are the lim-
ited drugs being clinically used for the treatment of LOX-mediated
diseases.7 MK-886 and MK-0591 are the other two most important
5-LOX inhibitors tested for the treatment of LOX mediated diseases
(Chart 1).
in zileuton and central core of MK-886 and MK-0591, indole moi-
ety was derivatized to procure new compounds for studying their
5-LOX inhibitory activities. In vitro enzyme immunoassay, stoichi-
ometry, association constants and molecular interactions of these
compounds for 5-LOX were investigated.
The reaction sequence adopted for the preparation of target
compounds is illustrated in Scheme 1. 2-(1H-Indol/5-methoxy-
1H-indole-3-yl)-2-oxoacetyl chloride (2) (93%) was obtained from
the reaction of 1H-indole/5-methoxy-1H-indole (1) with oxalyl
chloride at 0–5 °C using dry ether as the reaction medium. Treat-
ment of 2 with pyrrolidine and morpholine in presence of K2CO3
in acetonitrile gave respective compounds 3(a,b) and 4(a,b) in
71–83% yield. N-1 substitution of compounds 3 and 4 was achieved
through their reactions with various halides in presence of NaH in
dry acetonitrile to get target compounds 5(a–c) and 6(a,b) in 75–
94% yields. All the compounds were characterised by NMR spectra,
mass spectra and CHN analysis.
In vitro 5-LOX inhibitory activities of compounds 5 and 6 were
determined at 0.01, 0.1, 1 and 10 lM concentrations by using en-
zyme immuno assay kit according to the instructions provided
by the kit manufacturer.8 Compounds 5(a–c) and 6(a,b) exhibited
The uniqueness of indole in the biological systems, playing cru-
cial roles in the form of amino acid, growth hormone and alkaloids
and its presence at or near the catalytic/molecular recognition sites
of enzymes are the advantages for making indole as part of the
drug molecule. Similar to the benzothiophene fragment present
more than 50% inhibition of 5-LOX activity at 10 lM concentration.
The presence of morpholinyl moiety at the end of C-3 substituent
and cinnamyl and tosyl groups at N-1 in compounds 5c and 6b re-
sulted in appreciable inhibition of 5-LOX activity with IC50 5 and
1 lM, respectively (Table 1). Compounds with pyrolidinyl as a part
of C-3 substituent and tosyl, benzoyl and cinnamyl groups at N-1
(5a, 5b and 6a) showed significant inhibition of 5-LOX activity with
respective IC50 0.6, 1 and 1 lM. These compounds seem to be
⇑
Corresponding author. Tel.: +91 183 2258802 09x3495; fax: +91 183 2258819.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.068

1434
P. Singh, Pooja / Bioorg. Med. Chem. Lett. 23 (2013) 1433–1437
S
S
O
HO
NH2
O
N
N
N
COOH
N
COOH
S
CH3
Zileuton
Cl
Cl
MK-886
MK-0591
Chart 1. 5-LOX inhibitors.
O
Cl
R
R
i)
O
N
H
N
H
2
1
1a, 2a: R = H
1b, 2b: R = OCH
3
ii)
O
O
1
2
O
1
2
1
2
NR R
NR R
NR R
R
H CO
iii)
R = H
3
iii)
R = OCH
O
O
O
N
N
H
3, 4
3
N
3
3
R
R
5(a-c)
6(a-b)
1
1
2
2
3a: R=H, NR R =pyrolidinyl
1
1
1
2
3
5a: NR R =pyrolidinyl, R =tosyl
1
2
3b: R=H, NR R =morpholinyl
6a: NR R =pyrolidinyl,
2
2
3
3
5b: NR R =pyrolidinyl, R =benzoyl
1
1
2
2
3
4a: R=OCH , NR R =pyrolidinyl
R =cinnamyl
3
3
5c: NR R =morpholinyl, R =cinnamyl
1 2
4b: R=OCH , NR R =morpholinyl
6b: NR R =morpholinyl,
3
R =tosyl
1
2
3
Reaction conditions: i) (COCl) , dry ether
ii) dry ACN, K CO , NHR R
iii) NaH, dry ACN, R X
2
2
3
Scheme 1.
Table 2
Stoichiometry of the enzyme–compound complex and association constants
Table 1
5-LOX inhibitory activities of compounds 5 and 6
Compound m/z Free m/z
(b) À (a) Stoichiometry
Association
constant
(Ka)
Compound
% Inhibition of 5-LOX
0.1
IC50 (lM)
enzyme
(a)
Enzyme–
(enz:compd)
10
lM
1
lM
l
M
0.01 lM
compd
complex
(b)
5a
5b
5c
6a
6b
Zileuton
MK-0591
95.4
59.6
59.2
68.6
59.2
63.8
54.2
40.7
54.5
53.8
27.8
17.4
21.1
25.6
22.0
0.9
6.8
2.5
5.9
4.7
0.6
1
5
1
1
5a
5b
5c
6a
6b
Zileuton
86,957
86,957
86,957
86,957
86,957
86,957
89,641
88,335
88,203
89,274
88,812
89,656
2684
1378
1246
2317
1855
2699
1:7
1:4
1:3
1:6
1:4
1:9
1.2 Â 105
0.99 Â 105
0.53 Â 105
1.06 Â 105
1.96 Â 105
0.26 Â 104
3.7
5
better 5-LOX inhibitors than the clinically used zileuton (IC50
3.7 M) and MK-0591 (IC50
M).9 Therefore, the compounds with
appropriate combination of substituents at N-1 and C-3 of indole,
investigated here, seems to have sufficient potential for 5-LOX
inhibition.
The enzyme inhibition data of the test compounds led us to fur-
ther investigate the affinity and strength of association of these
compounds with 5-LOX. Since electrospray ionisation (ESI) mode
of mass spectrometer proved to be a versatile tool for studying
the enzyme–ligand interactions, the data corresponding to 5-LOX
inhibitory concentration of the compounds was supported with
mass spectra of 5-LOX in combination with compounds 5 and 6.
Mass spectrum of solution of compound 5a and 5-LOX in ACN–
H2O (7:3) was recorded in the +ve mode at source temperature
180 °C and collision energy 5–8 eV. Similar mass spectra were ob-
tained using ACN–H2O (1:9) as medium. Comparison of mass spec-
tra of enzyme recorded in ACN–H2O (7:3) and in pure water
l
5 l
indicated no change in the enzyme spectrum in ACN medium.
The compound–enzyme additive mass peak was observed at m/z
89641. Difference in the mass of compound–enzyme complex
and pure enzyme divided by the mass of the compound gave
5a–5-LOX stoichiometry 7:1 (Table 2, Fig. 1). Similarly, the com-
pound–enzyme additive mass peaks were observed for other com-
pounds and stoichiometries were calculated. In comparison to the
stoichiometry of zileuton with 5-LOX as calculated from mass
spectra shown in Figure 2, it was observed that the compounds un-
der present investigation have better affinity for 5-LOX. The asso-
ciation constants of enzyme–compound complexes were
calculated using the intensity of various mass peaks as equivalent
to the concentration of that species.10 In parallel with their enzyme
inhibitory concn (obtained from enzyme immunoassay), the mass
spectral technique also supported that all the five compounds
exhibited interactions with 5-LOX better than that of zileuton as

P. Singh, Pooja / Bioorg. Med. Chem. Lett. 23 (2013) 1433–1437
1435
Figure 1. Deconvoluted mass spectra of (a) 5a–5-LOX complex; (b) 5b–5-LOX complex; (c) 5c–5-LOX complex; (d) 6a–5-LOX complex; (e) 6b–5-LOX complex.
evident from their association constants with the enzyme (Table 2
and Table S1).
space was explored by the geometry optimization of the flexible li-
gand (rings are treated as rigid) in combination with the incremen-
tal construction of the ligand torsions. Thus, docking occurs
between the flexible ligand parts of the compound and enzyme.
The ligand orientation was determined by a shape scoring function
based on Ascore and the final positions were ranked by lowest
interaction energy values. H-bond and hydrophobic interactions
between the compound and enzyme were explored.
Dockings of the clinically used 5-lipoxygenase inhibitor, zileu-
ton in the active site of 5-LOX showed H-bond interactions with
amino acids A672, H372 and N554 through its hydroxy group, car-
bonyl group and amino group, respectively (Fig. 3). Docking of
compounds 5a and 5c in the active site of 5-LOX indicated H-bond
interactions with F177 and Q413 amino acid residues through the
oxygen of carbonyl group and morpholine ring (Fig. 4 and Fig. S3),
The results of above two experiments favouring the suitability
of the compounds for inhibition of 5-LOX incited to take molecular
modelling into consideration for looking into the nature of interac-
tions between the compound and active site amino acids of the en-
zyme. Compounds were built using the builder toolkit of the
software package ArgusLab 4.0.111 and energy minimized using
semi-empirical quantum mechanical method PM3. The crystal
coordinates of 5-LOX (pdb ID 3V99)12 were downloaded from pro-
tein data bank. Structure of this protein carries arachidonic acid as
a ligand in its binding site. In the molecule tree view, the active site
of the enzyme is defined as 15 Å around the ligand. The molecule to
be docked in the active site of the protein was pasted in the work
space carrying the structure of the enzyme. The conformational

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P. Singh, Pooja / Bioorg. Med. Chem. Lett. 23 (2013) 1433–1437
Figure 2. Deconvoluted mass spectra of (a) 5-LOX and (b) 5-LOX + zileuton.
Figure 4. Compound 5a (green) docked in the active site of 5-LOX. H-bonds between
carbonyl oxygen and amino acids Phe177 and Gln413 (pink) of the active site are
clearly visible. Arachidonic acid in the active site is shown in light blue colour.
Figure 3. Zileuton (green) docked in the active site of 5-LOX showing H-bonding
interactions with amino acids Ala672, His372 and Asn554 (shown in pink colour).
Arachidonic acid in the active site is shown in light blue colour.
while compound 6b interacted through its carbonyl oxygen and
morpholine oxygen with F177, Q413 and K409 amino acid residues
(Fig. S2). Compound 5b used its benzoyl carbonyl oxygen and
nitrogen of the indole ring to form H-bonds with Q557 and N554
amino acid residues (Fig. S1). Compound 6a showed H-bond inter-
actions of oxygen of carbonyl and methoxy group with amino acids
H367, H372 and N180 in the active site of enzyme 5-LOX (Fig. 5).
Besides hydrogen bond interactions, there are numerous non-
bonding interactions observed between the compound and the
amino acids in the active site. The indole ring in compounds 5a,
5b and 6b filled the cavity formed by amino acids I406, F169,
S171; H367, L607, Q363, Q557 and L607, F177, Q413, respectively.
N-1 benzoyl group and cinnamyl group in compounds 5b and 5c
showed a close approach to the amino acids A672, H372, H367,
N554 in the active site of 5-LOX, while the morpholine ring and
pyrrolidine ring in compounds 6b and 6a showed non-bonding
interactions with amino acids K409, S171, F169, I406 and A672,
H372, H367, Q363, respectively. Similarly the methoxy groups in
compounds 6b and 6a showed a close approach to amino acids
F177, Q413 and N180, L607, F177, respectively. It was observed
that compounds 5b and 6a interacted with the same amino acids
Figure 5. Compound 6a (green) docked in the active site of 5-LOX showing H-
bonding interactions through its carbonyl oxygen and methoxy oxygen with amino
acids Asn180, His372 and His367 (pink). Arachidonic acid in the active site is shown
in light blue colour.

P. Singh, Pooja / Bioorg. Med. Chem. Lett. 23 (2013) 1433–1437
1437
of 5-LOX (N554 and H372) where zileuton was bound. All these
References and notes
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Supplementary data
Supplementary data (experimental procedures, characterisation
of compounds, 1H NMR spectra of comopunds, calculation of asso-
ciation constants, Lipinski’s values and Figures S1–S3) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2012.12.068.