Lead modification: Amino acid appended indoles as highly effective 5-LOX inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.01.027

N-1 tosyl indoles carrying amino acid as a part of C-3 substituent are identified with considerable 5-LOX inhibitory activities. On the basis of enzyme inhibitory activities and log P, it is found that these compounds are more suitable to use as ester prodrugs. In addition to the significant K_a and K_i for 5-LOX, advantageously the compounds under present investigation do not affect the viability of the cell. The experimental results were also supported by molecular docking of compounds in the active site of 5-LOX.

Full text of DOI:10.1016/j.bmc.2014.01.027

Bioorganic & Medicinal Chemistry 22 (2014) 1642–1648
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lead modification: Amino acid appended indoles as highly effective
5-LOX inhibitors
⇑
Parteek Prasher, Pooja, Palwinder Singh
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:
N-1 tosyl indoles carrying amino acid as a part of C-3 substituent are identified with considerable 5-LOX
inhibitory activities. On the basis of enzyme inhibitory activities and logP, it is found that these com-
pounds are more suitable to use as ester prodrugs. In addition to the significant Ka and Ki for 5-LOX,
advantageously the compounds under present investigation do not affect the viability of the cell. The
experimental results were also supported by molecular docking of compounds in the active site of 5-LOX.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 21 November 2013
Revised 13 January 2014
Accepted 18 January 2014
Available online 29 January 2014
Keywords:
Indole
Amino acid
5-LOX inhibition
Molecular modeling
1. Introduction
agents,12 5-LOX inhibitors13 as well as in the form of agrochemi-
cals.14 Keeping in mind the biological acceptability of indole and
Symptomized with pain, increase in body temperature, redness,
swelling, loss of function etc.; a large group of human population
suffers from inflammatory diseases.1 Discovery of arachidonic acid
metabolic pathway,2 identification of enzymes participating
therein3 and recognition of inflammatory metabolites led to
systematic studies for development of anti-inflammatory drugs.4
Starting with stimulatory role of phospholipases to release
arachidonic acid from phospholipids, over-expression of cyclooxy-
genase-2 (COX-2) and lipoxygenase (LOX) enzymes cause more
than normal production of inflammatory prostaglandins and leu-
kotrienes.5 Hence these enzymes are the targets of majority of
anti-inflammatory drugs. Irrespective of the long journey of anti-
inflammatory drugs, from aspirin to COXIBS4,6—targeting COX-2
and zileuton, MK-886 and MK-095—targeting LOX,7 still the safety
(devoid of side effects) of the drug/s is a primary issue.8 This neces-
sitates the search for such compounds which may prove as more
efficacious and safer drugs. In continuation of our previous report,9
another set of compounds is designed, synthesized and evaluated
for inhibition of catalytic activity of various enzymes of arachi-
donic acid metabolic pathway.
amino acids; herein, compounds 2–3 (Chart 1) were obtained by
modifying the previous compound 1.9 While in compound 1, tosyl
group was optimized, here, presence of amino acid as part of C-3
substituent led to the development of highly potent 5-LOX
inhibitors.
2. Result and discussion
2.1. Chemistry
The sequence of steps for the synthesis of target compounds is
illustrated in Schemes 1 and 2. Oxalyl chloride was added to solu-
amino acid
ester
amino acid
O
N
O
O
O
O
O
N
S
N
S
N
S
O
O
O
O
O
O
Structural and functional diversity of naturally occurring indole
has made it a privileged pharmacophore in drug development
where indole based natural and synthetic compounds find myriad
properties as anti-microbial,10 anti-inflammatory,11 anti-cancer
CH3
CH3
CH3
3
2
1
2-3: amino acid/ester; a) glycine, b) tryptophane, c) histidine, d) glutamic acid, e) proline
⇑
Corresponding author. Tel.: +91 183 2258802-09x3495; fax: +91 183 2258820.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
Chart 1. Based on previous compound 1, design of new molecules 2 and 3.
http://dx.doi.org/10.1016/j.bmc.2014.01.027
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

P. Prasher et al. / Bioorg. Med. Chem. 22 (2014) 1642–1648
1643
N
H
i
O
O
O
H
N
Cl
H
N
NH
iii
COOMe
ii
O
C
O
H
N
H
4
O
N
H
N
H
COOMe
5a
5b
vi
iv
v
N
O
O
H
NH
N
N
C
O
H
COOCH3
H
OMe
O
O
N
O
N
H
H
COOMe
H
N
5e
5c
C
O
N
H
COOCH3
5d
Reaction conditions:
i) (COCl)2 , dry ether, 0 o
C
ii) Dry ACN, K2CO3, glycine methyl esterHCl, 0-5 o
C
iii) Dry ACN, K2CO3, L-tryptophan methyl esterHCl, 0-5 o
iv) Dry ACN, K2CO3, L-histidine methyl esterHCl, 0-5 o
v) Dry ACN, K2CO3, L-glutamic methyl diesterHCl, 0-5 o
vi) Dry ACN, K2CO3, L-proline methyl esterHCl, 0-5 o
C
C
C
C
Scheme 1.
tion of indole in dry ether at 0 °C and resulting yellow colored solid
(4) was filtered out from the reaction mixture. Compound 4, taken
in dry acetonitrile was treated with L-amino acid ester hydrochlo-
nM and 10 nM, respectively. Moreover, compound 3d also exhib-
ited significant 5-LOX inhibitory activity with IC50 97 nM. Com-
pounds 3a and 3b were found to be showing inhibition of 5-LOX
enzymatic activity comparable to that of zileuton16 and MK
0591.17
rides in presence of K2CO3. After stirring the reaction mixture for
25–30 min, it was purified with column chromatography to get
3-substituted indole 5a–e in 70–80% yield (Scheme 1). Taken in
dry acetonitrile, compounds 5a–e were treated with p-tolylsulfo-
nyl chloride in presence of NaH keeping reaction temperature 0–
5 °C. After usual work up, compounds 2a–e were procured in 80–
85% yield. Further, hydrolysis of compounds 2a–e with NaOH pro-
vided compounds 3a–e in quantitative yield (Scheme 2). All the
compounds were characterized with NMR and high resolution
mass spectra (Supporting information).
Therefore, the trends of 5-LOX inhibitory activities of com-
pounds 2, 3 and 5 indicate that compounds 3 are more suitable
for their use as 5-LOX inhibitors. Since a proper balance between
lipophilicity and hydrophilicity is one of the primary criteria to ad-
judge the suitability of compound for medicinal purpose, partition
coefficient of compounds 2, 3 and 5 was calculated (ClogP).18 LogP
for compounds 5 was found to be 0.3–1.5 (Table 2) which is quite
low. In presence of tosyl group at N-1 position of compounds 5, the
resulting compounds 2 have desirable logP, ꢀ2. Experimental logP
of compounds 2 (determined by octanol–water partition) was
quite close to the theoretical values. However, compounds 3 exhib-
ited quite low partition coefficient. Hence, it may be recommended
to use compound 2 (instead of compound 3) as 5-LOX inhibitors
which may undergo hydrolysis under the action of esterase (pres-
ent in sufficient amount in the cell) to form compounds 3 when
administered to the biological system. In vitro hydrolysis of com-
pounds 2 to 3 using HEPES buffer as medium was successfully per-
formed. Interestingly, 5-LOX inhibitory activities of compounds 2,
checked through enzyme immunoassay performed in presence of
pig liver esterase, were found to be quite close to those of com-
pounds 3 (Table 1) indicating the in situ enzymatic (esterase)
hydrolysis of compounds 2 to 3. It seems that substituent at N-1
position of indole might be responsible for maintaining appropri-
ate hydrophilic-lipophilic balance in the compound. However,
more information about the role of different substituents present
on indole moiety of compounds 2–3 was obtained from the results
of docking studies of these compounds in the active site of 5-LOX
enzyme.
2.2. Enzyme immunoassays
2.2.1. 5-LOX inhibitory activities
In vitro 5-LOX inhibitory activities of compounds 2, 3 and 5
were checked at 0.01 lM, 0.1 lM, 1 lM, 10 lM and 100 lM con-
centrations using enzyme immunoassay kits.15 The results of these
experiments in terms of percentage inhibition of 5-LOX activity at
different concn of the compounds and their respective IC50 are gi-
ven in Table 1. Although compounds under present investigation
may not have same mode of action as zileuton and MK 0591 but
5-LOX inhibitory data of these two drugs was taken for compari-
son. All the compounds except 5a–e exhibited appreciable inhibi-
tion of 5-LOX activity. Comparison of enzyme immunoassay
results of compounds 5 with 2 and 3 indicated that a substituent
at N-1 position of indole is essential for inhibition of 5-LOX enzy-
matic activity. Amongst compounds 2, it was observed that 2a
and 2b, carrying respectively glycine and tryptophan functionality
were most active with IC50 9.7 nM and 52 nM. Compounds 3, the
corresponding acids of esters 2, exhibited considerably higher 5-
LOX inhibitory activity than their ester analogues except in case
of compound 3e. Here again, compounds 3a and 3b with glycine
and tryptophan residue at the end of C-3 substituent were identi-
fied as the most active amongst the compounds under present
investigation. IC50 of compounds 3a and 3b was found to be 8.6
2.2.2. Enzyme immunoassays with COX-2 and PLA2
In order to check if these compounds also inhibit the catalytic
activity of other enzymes of arachidonic acid pathway, their en-
zyme immunoassays with COX-2, COX-1 and PLA2 were per-

1644
P. Prasher et al. / Bioorg. Med. Chem. 22 (2014) 1642–1648
O
O
H
N
H
N
COOMe
COOH
O
O
N
S
N
S
i
ii
5a
O
O
O
O
CH3
2a
CH3
3a
O
H
O
NH
H
N
NH
H
N
C
H
C
O
O
N
S
COOH
N
S
COOMe
ii
i
O
O
5b
O
O
CH3
3b
CH3
2b
N
O
H
N
N
N
H
O
H
C
H
COOH
N
H
N
O
C
N
S
H
O
N
COOMe
O
O
S
ii
i
O
O
5c
CH3
3c
2c
CH3
COOH
H
O
H
N
COOCH3
O
C
H
O
N
N
S
COOH
C
H
O
O
O
N
COOCH3
ii
S
i
O
O
5d
CH3
O
3d
CH3
2d
O
N
N
O
OH
O
O
N
S
OMe
O
N
S
ii
i
5e
O
O
O
O
CH3
3e
2e
CH3
Reaction conditions:
i) NaH, Dry ACN , p-tolylsulphonyl chloride, 0-5 oC; ii) 1N NaOH, acetone-water
Scheme 2.
formed.19 However, we did not observe change in the catalytic
activity of these three enzymes in presence of compounds 2, 3
and 5. Hence, similar to the 5-LOX selectivity of zileuton,20
amongst other enzymes of arachidonic acid pathway, compounds
2 and 3 also target 5-LOX only and do not seem to be the frequent
hitters. The selective 5-LOX inhibitory activity of zileuton for the
treatment of disorders like asthma is well documented.20
As it is apparent from the data given in Table 3, compounds 2
and 3 exhibited appreciable Ka for 5-LOX. Similar to the results of
enzyme immunoassay, compounds 2a and 3a with their respective
Ka 2.32 ꢃ 106 and 2.07 ꢃ 106 showed significant association with
5-LOX. Moreover, the Ki (inhibitor concentration required to pro-
duce half maximum inhibition) of compounds 2 and 3 for 5-LOX
(Table 3) were also calculated with Eq. (2).23
Ki ¼ IC50=1 þ ½Lꢂ=Kd
ð2Þ
2.2.3. Ka (Association constant) and Ki (inhibitor constant) of
compounds 2 and 3 for 5-LOX
Based on the results of 5-LOX enzyme immunoassay, interactions
of compounds 2 and 3 with 5-LOX were investigated with the help of
UV–visible spectral studies21 and the association constant of com-
pounds 2 and 3 with 5-LOX were calculated using Eq. 1.22
2.2.4. Cell viability assay
To check the toxicity of the compounds towards mammalian
cells, the cell viability assay was performed with compounds 2a
and 3a. Mammalian HeLa cells were treated with compounds 2a
and 3a and analyzed the culture using MTT assay at different time
points. As it is evident from Figure 1, there was no change in the
1=Af ꢁ Aobs ¼ 1=Af ꢁ Afc þ 1=Ka½Af ꢁ Afcꢂ½Lꢂ
ð1Þ
where Af = absorbance of free host, Aobs = absorbance observed,
growth of HeLa cells even with 100
2a and 3a.
lg/ml (ꢀ0.25 M) of compounds
Afc = absorbance at saturation

P. Prasher et al. / Bioorg. Med. Chem. 22 (2014) 1642–1648
1645
M)
Table 1
5-LOX inhibitory activities of compounds 2, 3 and 5
*
#
Compd
% Inhibition of 5-LOX at compound concn
IC50
(l
M)
IC50 (l
100
l
M
10
lM
1
lM
0.1
l
M
0.01 lM
5a
5b
5c
5d
5e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
70
65
65
55
60.6
98
80.4
63.8
65
72
97.5
85
55.5
49.8
45.5
51.2
59.8
85
47.8
43.4
40.8
44.8
40.2
67.67
62.70
45.3
51.8
54.5
78.4
67.3
48.7
59.5
53.5
46.2
41.1
37.7
40
25.5
51.5
55.3
40.5
35.19
48.7
63.5
56.5
28.5
50.5
45
40.0
35.0
30.0
36
22.5
50
45.5
30
18.46
38.50
57.4
51
3.5
—
—
8.33
5.46
0.0097
0.052
4.33
—
—
—
—
—
0.0080
0.014
2.98
0.13
0.75
—
75.5
58.18
60.33
69.33
91.2
81.5
55.5
0.90
0.33
0.0086
0.010
2.80
0.097
0.60
—
—
—
—
64.7
90.6
74.3
20
42.0
27.3
87.59
68.8
Zileuton
MK 0591
0.3
—
—
1.6#
*
0.5 of two experiments; #value in nM.
IC50 of compounds 2 checked in presence of pig liver esterase.
#
Table 2
2.3. Docking studies
C logP of compounds 2, 3 and 5
Since the compounds under present investigation showed
appreciable inhibition of the catalytic activity of 5-LOX, it was
planned to scrutinize the mode of interactions of compounds 2
and 3 with amino acids in the active site of 5-LOX. For this purpose,
molecular docking of these compounds in the active site of the en-
zyme was performed. Using ArgusLab 4.0.1,24 compounds 2 and 3
were docked in the active site of 5-LOX (pdb ID 3v99).25 It was ob-
served that most of these compounds exhibit H-bond interactions
with active site amino acids of 5-LOX but with significant differ-
ence in their respective docking score (Table 4). Compound 2a
showed H-bond interaction with Q554 and Q557 through its
COOMe group and indole N (Fig. 2), respectively while compound
2b through its ketonic O and amidic O interacted with F177,
A410 and N413 (Fig. S33). However, docking of compound 3a in
the active site of 5-LOX showed that this compound interact more
effectively with the active site amino acids in comparison to the
interactions of compound 2a with the amino acids of same en-
zyme. Compound 3a exhibited H-bond interactions with Q554,
Q557, V604 and N554 through sulfonyl oxygen, indole ring N, ami-
dic O and carboxyl O, respectively (Figs. 3–5). Compound 3b also
interacted with active site amino acids through its sulfonyl O and
carboxyl O (Fig. 6). It seems that similar to the mode of action of
indomethacin, the most potent compounds in the present studies
also interact with 5-LOX through their carboxyl functionality. For
comparing the molecular interactions of compounds 2 and 3 with
Compd ClogP Compd ClogP Experimental logP Compd ClogP
5a
5b
5c
5d
5e
0.579
1.52
0.323
0.661
1.213
2a
2b
2c
2d
2e
2.003
2.220
2.020
2.010
2.061
2.21
2.34
1.98
2.12
2.19
3a
3b
3c
3d
3e
ꢁ0.474
1.872
ꢁ0.05
ꢁ0.295
0.565
Table 3
Association constant (Ka) of compound 2 and 3 with 5-LOX
Compd
Ka (Mꢁ1
)
Ki (lM)
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
2.32 ꢃ 106
6.67 ꢃ 105
1.01 ꢃ 104
1.06 ꢃ 105
1.2 ꢃ 105
2.07 ꢃ 106
3.6 ꢃ 105
3.7 ꢃ 104
1.1 ꢃ 105
2.6 ꢃ 105
0.0095
0.037
4.16
0.82
0.32
0.0084
0.0099
2.53
0.095
0.51
Table 4
Docking score of compounds 2 and 3
Compd
Docking score (K cal/mol)
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
Zileuton
MK886
ꢁ20.0
ꢁ9.0
ꢁ11.5
ꢁ13.0
ꢁ14.2
ꢁ25.4
ꢁ24.2
ꢁ13.7
ꢁ17.0
ꢁ13.8
ꢁ9.18
ꢁ9.96
Figure 1. Growth of HeLa cells was not affected by compounds 2a and 3a.

1646
P. Prasher et al. / Bioorg. Med. Chem. 22 (2014) 1642–1648
Figure 2. Compound 2a docked in the active site of 5-LOX. Hs’ are omitted for
clarity.
Figure 5. Crystal coordinates of 5-LOX in association with compound 3a (generated
in pymol). Dotted lines indicate H-bond interactions of compound 3a with amino
acid residues.
Figure 3. Compound 3a docked in the active site of 5-LOX. (pdb ID 3V99).
Figure 6. Crystal coordinates of 5-LOX in association with compound 3b (green)
(generated in Pymol). Dotted lines indicate H-bond interactions between the
compound and amino acids.
that of zileuton and MK886 in the active site of 5-LOX, these two
standard drugs were also docked in the active site of 5-LOX. How-
ever, H-bond interactions of zileuton (Fig. S41) and MK886
(Fig. S42) as well as their respective docking scores were less in
comparison to compounds 2a, 3a and 3b. Therefore, supporting
the experimental results, the molecular docking of the compounds
Figure 4. Compound 3a docked in the active site of 5-LOX. Compound 3a (ball and
stick model) is placed in the same pocket where AA (red wire) is present.

P. Prasher et al. / Bioorg. Med. Chem. 22 (2014) 1642–1648
1647
in the active site of 5-LOX provided an insight into the mode of
4.5. Cell viability assay
interactions between the compounds and amino acids.
Effect of the compounds 2a and 3a on the growth of cells was
monitored using MTT assay. Performing the experiment in tripli-
cate, HeLa cells (4000 cells/well) were incubated in a 96-well plate
3. Conclusions
in the presence of 100
ume of 200 L at 37 °C in a humidified chamber. Cells with solvent
only (DMSO) were taken as a control. After certain intervals, 20
of MTT solution (5 mg/ml in PBS) was added to each well and incu-
bated for another 4 h. After removing the supernatant at the end of
lg/ml of compound 2a and 3a with final vol-
In conclusion, improving over the previous compound, amino
acid appended indoles were identified with considerable increase
in their biological activity. Two compounds exhibited IC50 for 5-
LOX in nm range. Physico-chemical parameters and in vitro inves-
tigations indicate the use of compounds 2 as prodrugs of their
more active form 3. Appreciable Ka and Ki of compounds 2a and
3a for 5-LOX, supported by molecular docking results and their
non-toxicity to mammalian cells indicate the suitability of these
compounds for further investigations. More compounds of this cat-
egory by introducing two/three amino acids at C-3 position of in-
dole will also be explored for their 5-LOX inhibitory potential.
l
lL
4 h, the resultant formazan crystals were dissolved in 200
lL
DMSO and absorbance (A) was measured at 570 nm by a micro-
plate reader. The percentage viability of the cells was calculated
by using the following equation.
Percentage of cell viability ¼ ðabsorbance of test samples=
absorbance of the control sampleÞ ꢃ 100:
4. Experimental Procedure
4.6. Docking Procedure
4.1. 5-LOX inhibtory activities
For 5-LOX inhibition studies, solutions of compounds at
Compounds were built using the builder toolkit of the software
package ArgusLab 4.0.1 and energy minimized using semi-empiri-
cal quantum mechanical method PM3. The crystal coordinates of
5-lipoxygenase (pdb ID 3V99) were downloaded from protein data
bank, carrying arachidonic acid as ligand in its binding site. 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 dock-
ing programme implements an efficient grid based docking algo-
rithm which approximates an exhaustive search within the free
volume of the binding site cavity. The conformational space was
explored by the geometry optimization of the flexible ligand (rings
were treated as rigid) in combination with the incremental con-
struction 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 (docking score in Kcal/mol). H-bond and hydropho-
bic interactions between the compound and enzyme were
explored.
0.01
prepared in DMSO. 10
centrations was added to 90
l
M, 0.1
l
M, 1
l
M, 10
L of each compound from the above con-
L solution (in assay buffer) of 5-
lM and 100 lM concentrations were
l
l
LOX enzyme taken in the wells of a 96-well plate. Each compound
was tested in duplicate and the average of two values with devia-
tion <5% was taken for calculation. Two wells were taken as blanks
(assay buffer + AA) and four wells were taken as positive controls
(enzyme in assay buffer + AA). The reaction was initiated by the
addition of 10
lL of the substrate (AA). After shaking the 96-well
plate on a shaker for 5 min, 100
lL of the chromogen (developing
reagent) was added to each well. The plate was again shaken for
5 min and read at 490 nm in a microplate scanning spectropho-
tometer. Percent 5-LOX inhibitory activity was determined using
the mean of the two values for each sample from the calculations
given below:Lipoxygenase activity (lmol/min/ml) = [A500/min/
9.47 mMꢁ1] ꢃ [0.21 ml/0.09 ml] ꢃ sample dilutionwhere A500/
min = A500 (sample)/min ꢁ A500 (blank)/5 min, 9.47 mMꢁ1 = -
Extinction coefficient of chromogen, 0.21 ml = Total volume of
the solution in each well and 0.09 ml = Volume of the enzyme solu-
tion used.Percent 5-LOX inhibition = [{L.A. (P.C.) ꢁ L.A. (I.)}/L.A.
(P.C.)] ꢃ 100
Conflict of interest
L.A. (P.C.) ꢁ Lipoxygenase Activity of positive control, L.A.
(I.) ꢁ Lipoxygenase Activity of inhibitor.
The authors declare they have no conflict of interests.
Acknowledgments
IC50 values were determined from the graph between Percent
Inhibition versus concn of inhibitor using KaleidaGraph 3.5.
Financial assistance from CSIR, New Delhi and DST, New Delhi is
gratefully acknowledged. P.P. thanks CSIR for junior research
fellowship.
4.2. 5-LOX inhibtory activities of compounds 2 in presence of
pig liver esterase
This assay was performed through the same procedure as above
except that compound 2 was treated with pig liver esterase (ex-
tracted by following reported procedure) before it is added to the
5-LOX enzyme.
Supplementary data
Supplementary data (experimental data, NMR spectra, mass
spectra of compounds and docking structures) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2014.01.027.
4.3. COX-1/2 and PLA2 inhibitory activities
COX-1/2 and PLA2 enzyme inhibitory activities of compounds 2
and 3 were checked as per the protocol given with each enzyme
kit.19
References and notes
1. W.H.O. data sheet 2012. www.who.int.
2. (a) Sraer, J.; Rigaud, M.; Bens, M.; Rabinovitch, H.; Ardaillou, R. J. Biol. Chem.
1983, 258, 4325; (b) Borgeat, P. J. Med. Chem. 1981, 24, 121. and references
therein.
4.4. UV–visible spectral studies for calculating Ka
3. (a) Zeldin, D. C. J. Biol. Chem. 2001, 276, 31059. and references therein.
4. (a) Blobaum, Anna L.; Marnette, Lawrence J. J. Med. Chem. 2007, 50, 1425; (b)
Singh, P.; Mittal, A. Mini Rev. Med. Chem. 2008, 8, 73.
Same protocol was used as did previously for studying com-
pound–DHFR interactions.20

1648
P. Prasher et al. / Bioorg. Med. Chem. 22 (2014) 1642–1648
5. (a) Haeggstrom, J. Z.; Funk, C. D. Chem. Rev. 2011, 111, 5866; (b) Nakamura, M.;
Shimizu, T. Chem. Rev. 2011, 111, 6231; (c) Samuelsson, B. Angew. Chem., Int. Ed.
Engl. 1982, 21, 902.
14. Andreani, A.; Rambaldi, M. J. Heterocycl. Chem. 1988, 25, 1519.
15. 5-LOX inhibitor screening assay kit, Item No. 760700. Cayman Chemical Co.
16. Carter, G. W.; Young, P. R.; Albert, D. H.; Bouska, J.; Dyer, R.; Bell, R. L.;
Summers, J. B.; Brooks, D. W. J. Pharmacol. Exp. Ther. 1991, 256, 929.
17. Brideau, C.; Chan, C.; Charleson, S.; Denis, D.; Evans, J. F.; Ford-Hutchinson, A.
W.; Fortin, R.; Gillard, J. W.; Guay, J.; Guévremont, D., et al Can. J. Physiol.
Pharmacol. 1992, 70, 799.
6. Green, G. A. Clin. Cornstone 2001, 3, 50.
7. Pergola, C.; Werz, O. Expert Opin. Ther. Pat. 2010, 20, 355.
8. (a) Dogne, J.-M.; Supuran, C. T.; Pratico, D. J. Med. Chem. 2005, 48, 2251; (b)
Leite-Pereira, C.; Nunes, C.; Lima, J. L. F. C.; Reis, S.; Lucio, M. J. Phys. Chem. 2012,
116, 13608; (c) Food and Drug Administration, News release, September 30,
2004 and April 7, 2005.
9. Singh, P.; Pooja Bioorg. Med. Chem. Lett. 2013, 23, 1433. and references therein.
10. (a) Farghalay, A. H.; Rahman, A. J. Chin. Chem. Soc. 2004, 51, 147; (b) Benkli, K.;
Demirayak, S.; Karaburun, G. N.; Kiraz, N.; Iscan, G.; Ucucu, U. Ind. J. Chem.
2004, 43B, 174; (c) Hiremath, D. M.; Hiremath, S. C.; Yadawe, M. S. Res. J. Pharm.
Biol. Chem. Sci. 2012, 3, 969.
11. Chandra, T.; Garg, N.; Kumar, A. Int. J. Chem. Tech. Res. 2010, 2, 762.
12. Sharma, V.; Kumar, P.; Pathak, D. J. Heterocycl. Chem. 2010, 47, 491.
13. (a) Zheng, M.; Zheng, M.; Ye, D.; Deng, Y.; Qiu, S.; Luo, X.; Chen, K.; Liu, H.;
Jiang, H. Bioorg. Med. Chem. Lett. 2007, 17, 2414; (b) Landwehr, J.; George, S.;
Karg, E.-M.; Poeckel, D.; Steinheilber, D.; Troschuetz, R.; Werz, O. J. Med. Chem.
2006, 49, 4327.
18. www.molinspiration.com.
19. Cayman Chemical Co., Cat. No. 560131 (COX-1/2) and Cat no. 765021 (cPLA2).
20. Berger, W.; De Chandt, M. T. M.; Cairns, C. B. Int. J. Clin. Pract. 2007, 61, 663.
21. For UV–visible spectral studies, same procedure was followed as reported for
similar studies with DHFR. Singh, P.; Kaur, M.; Sachdeva, S. J. Med. Chem. 2012,
55, 6381.
22. Hildebrand, J. H.; Benesi, H. A. J. Am. Chem. Soc. 1949, 71, 2703.
23. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmcol. 1973, 22, 413.
24. ArgusLab 4.0.1, M. A. Thompson, Planaria Software LLC, Seattle, WA 98155.
25. Gilbert, N. C.; Rui, Z.; Neau, D. B.; Waight, M. T.; Bartlett, S. G.; Boeglin, W. E.;
Brash, A. R.; Newcomer, M. E. FASEB J. 2012, 26, 3222.