Design, synthesis and identification of novel coumaperine derivatives for inhibition of human 5-LOX: Antioxidant, pseudoperoxidase and docking studies

Article abstract of DOI:10.1016/j.bmc.2018.12.043

5-Lipoxygenase (5-LOX) is a key enzyme involved in the biosynthesis of pro-inflammatory leukotrienes, leading to asthma. Developing potent 5-LOX inhibitors especially, natural product based ones, are highly attractive. Coumaperine, a natural product found in white pepper and its derivatives were herein developed as 5-LOX inhibitors. We have synthesized twenty four derivatives, characterized and evaluated their 5-LOX inhibition potential. Coumaperine derivatives substituted with multiple hydroxy and multiple methoxy groups exhibited best 5-LOX inhibition. CP-209, a catechol type dihydroxyl derivative and CP-262-F2, a vicinal trihydroxyl derivative exhibited, 82.7% and 82.5% inhibition of 5-LOX respectively at 20 μM. Their IC₅₀ values are 2.1 ± 0.2 μM and 2.3 ± 0.2 μM respectively, and are comparable to zileuton, IC₅₀ = 1.4 ± 0.2 μM. CP-155, a methylenedioxy derivative (a natural product) and CP-194, a 2,4,6-trimethoxy derivative showed 76.0% and 77.1% inhibition of 5-LOX respectively at 20 μM. Antioxidant study revealed that CP-209 and 262-F2 (at 20 μM) scavenged DPPH radical by 76.8% and 71.3% respectively. On the other hand, CP-155 and 194 showed very poor DPPH radical scavenging activity. Pseudo peroxidase assay confirmed that the mode of action of CP-209 and 262-F2 were by redox process, similar to zileuton, affecting the oxidation state of the metal ion in the enzyme. On the contrary, CP-155 and 194 probably act through some other mechanism which does not involve the disruption of the oxidation state of the metal in the enzyme. Molecular docking of CP-155 and 194 to the active site of 5-LOX and binding energy calculation suggested that they are non-competitive inhibitors. The In-Silico ADME/TOX analysis shows the active compounds (CP-155, 194, 209 and 262-F2) are with good drug likeliness and reduced toxicity compared to existing drug. These studies indicate that there is a great potential for coumaperine derivatives to be developed as anti-inflammatory drug.

Full text of DOI:10.1016/j.bmc.2018.12.043

Accepted Manuscript
Design, Synthesis and Identification of Novel Coumaperine Derivatives for In-
hibition of Human 5-LOX: Antioxidant, Pseudoperoxidase and Docking Studies
Subramani Muthuraman, Shweta Sinha, C.S. Vasavi, Kamran Manzoor Waidha,
Biswarup Basu, Punnagai Munussami, M.M. Balamurali, Mukesh Doble,
Rajendran Saravana Kumar
PII:
S0968-0896(18)31716-4
https://doi.org/10.1016/j.bmc.2018.12.043
BMC 14692
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
5 October 2018
14 December 2018
31 December 2018
Please cite this article as: Muthuraman, S., Sinha, S., Vasavi, C.S., Waidha, K.M., Basu, B., Munussami, P.,
Balamurali, M.M., Doble, M., Kumar, R.S., Design, Synthesis and Identification of Novel Coumaperine Derivatives
for Inhibition of Human 5-LOX: Antioxidant, Pseudoperoxidase and Docking Studies, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.12.043
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1
Design, Synthesis and Identification of Novel Coumaperine Derivatives for Inhibition of
Human 5-LOX: Antioxidant, Pseudoperoxidase and Docking Studies
a
bc$
d
e
f
Subramani Muthuraman , Shweta Sinha , Vasavi C. S , Kamran Manzoor Waidha , Biswarup Basu ,
g
a
*c
*a
Punnagai Munussami , Balamurali M. M , Mukesh Doble , Rajendran Saravana Kumar
a
Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology, Chennai-600127,
Tamilnadu, India. E-mail: sar.org@gmail.com and saravanakumar.r@vit.ac.in
b
Department of Chemistry, Vellore Institute of Technology, Vellore, Tamilnadu 632014, India.
c
Department of Biotechnology, Indian Institute of Technology, Madras, Tamilnadu 600036, India. E-
mail: mukeshd@iitm.ac.in.
d
Bioinformatics Division, School of Biosciences and Technology, VIT University, Vellore, Tamilnadu 632
0
14, India.
e
Amity Institute of Biotechnology, Amity University Uttar Pradesh,sector-125,Noida-201303,India.
f
Department of Neuroendocrinology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road,
Kolkata 700026,India
g
Center for Computational Natural Sciences and Bioinformatics, International Institute of Information
Technology, Gachibowli, Hyderabad - 500 032, India.
$
Equally contributed with the first author for this work.
*
1
Corresponding authors
) Dr. Rajendran Saravana Kumar, Ph.D., Assistant Professor, School of Advanced Sciences,
Vellore Institute of Technology, Chennai Campus, Vandalur-Kelambakkam Road, Chennai-
6
3
2
00127 Tamilnadu, India. Email: sar.org@gmail.com. Phone: +91-44-39931479 Fax: 91-44-
9932555.
) Dr. Mukesh Doble, Ph.D., Professor, Department of Biotechnology, Indian Institute of
Technology, Madras, Tamilnadu 600036, India.

2
Abstract
-Lipoxygenase (5-LOX) is a key enzyme involved in the biosynthesis of pro-inflammatory
5
leukotrienes, leading to asthma. Developing potent 5-LOX inhibitors especially, natural product
based ones, are highly attractive. Coumaperine, a natural product found in white pepper and its
derivatives were herein developed as 5-LOX inhibitors. We have synthesized twenty four
derivatives, characterized and evaluated their 5-LOX inhibition potential. Coumaperine
derivatives substituted with multiple hydroxy and multiple methoxy groups exhibited best 5-
LOX inhibition. CP-209, a catechol type dihydroxyl derivative and CP-262-F2, a vicinal
trihydroxyl derivative exhibited, 82.7% and 82.5% inhibition of 5-LOX respectively at 20 µM.
Their IC values are 2.1 ± 0.2 µM and 2.3 ± 0.2 µM respectively, and are comparable to
5
0
zileuton, IC = 1.4 ± 0.2 µM. CP-155, a methylenedioxy derivative (a natural product) and CP-
5
0
1
94, a 2,4,6-trimethoxy derivative showed 76.0% and 77.1% inhibition of 5-LOX respectively at
0 µM. Antioxidant study revealed that CP-209 and 262-F2 (at 20 µM) scavenged DPPH radical
2
by 76.8% and 71.3% respectively. On the other hand, CP-155 and 194 showed very poor DPPH
radical scavenging activity. Pseudo peroxidase assay confirmed that the mode of action of CP-
2
09 and 262-F2 were by redox process, similar to zileuton, affecting the oxidation state of the
metal ion in the enzyme. On the contrary, CP-155 and 194 probably act through some other
mechanism which does not involve the disruption of the oxidation state of the metal in the
enzyme. Molecular docking of CP-155 and 194 to the active site of 5-LOX and binding energy
calculation suggested that they are non-competitive inhibitors. The In-Silico ADME/TOX
analysis shows the active compounds (CP-155, 194, 209 and 262-F2) are with good drug
likeliness and reduced toxicity compared to existing drug. These studies indicate that there is a
great potential for coumaperine derivatives to be developed as anti-inflammatory drug.

3
1
. Introduction
The enzymatic pathway of 5-LOX was described in 1976 and subsequent studies have revealed
1
,2
the pathophysiological role of 5-LOX in respiratory and cardiovascular diseases. 5-LOX is a
non-heme metalloenzyme possessing iron at the active site. In the resting state, iron is in ferrous
form and in the active state in ferric form. Hydroperoxidation of lipids due to physiological stress
may activate 5-LOX. It is shown that in the cascade of 5-LOX pathway, the activated enzyme in
2
+
the cytoplasm is translocated to nuclear membrane upon Ca binding and/or by phosphorylation.
With the aid activating protein, FLAP, 5-LOX at the nuclear membrane catalyzes the
hydroperoxidation of arachidonic acid (a polyunsaturated acid, secreted in excess at the nuclear
membrane as a consequence of activation of cytosolic phospholipase A2 (cPLA )) and
2
subsequently to pro-inflammatory leukotrienes (LTs). Leukotrienes are known to be lipid
2
-5
mediators for inflammatory response with major roles in cardiovascular diseases. Results from
recent studies also suggest the role of leukotrienes in prostate cancer, osteoporosis and in certain
1
,2,6
forms of leukemia.
5-LOX is validated as a potential drug target for treating asthma,
rheumatoid arthritis, osteoarthritis, allergic, cardiovascular diseases and certain types of
5
,7
cancer. For more than three decades considerable progress have been made towards finding
more potent 5-LOX inhibitors. Several natural products, their analogs and new scaffolds have
been identified as potent 5-LOX inhibitors, to name a few, nordihydroguaretic acid (a
polyphenolic compound isolated from creosote plant), curcumin (a polyphenolic compound
found in turmeric), eugenol (a polyphenolic compound found in clove) and quercetin (a flavone
8
9
found in garlic), Hyperforin (a phytochemical isolated from Hypericum perforatum), zileuton,
1
0
11
ICI207968, ICID2138, 3-tridecyl-4,5-dimethoxybenzene-1,2-diol and Licofelone (Scheme
1
2
1
). Based on the inhibitory mode of action, the 5-LOX inhibitors are categorized into four

4
2
types, (i) redox inhibitors (redox-active compounds interrupts the redox cycle of the enzyme),
(ii) iron-ligand inhibitors with weak redox active properties (inhibit by chelating to active site
iron), (iii) non-redox inhibitors (compete with the substrate, arachidonic acid at the active site),
and (iv) allosteric inhibitors (inhibition by binding other than active site). Despite considerable
efforts made towards the development of efficient drugs that target 5-LOX enzyme, zileuton
remains the only clinically approved 5-LOX inhibitor in the treatment of asthma but its use is
limited due to severe side effects such as, weak potency, liver toxicity, and an unfavorable
1
,13
pharmacokinetic profile with a short half-life. Therefore, there is a strong need for the
development of safer and more potent 5-LOX inhibitors.
From the prehistoric times, plant based natural products are excellent source of therapeutic
medicines, species of the genus Piper are among the important class of natural product used in
various system of medicine, Ayurveda, Siddha and Unani for treating several illness such as,
joint pain, liver problem, lung diseases and hernia in traditional herbal medicines in India and in
1
4
other East Asian countries. Black and long pepper occupies special status in Indian traditional
medicines. They have been used for treating various inflammatory diseases such as asthma,
tuberculosis, bronchoconstriction, inflammation, allergic and rheumatoid arthritis. Black and
long pepper, their extracts and their bioactive alkaloid, piperine (Scheme 1) have been shown to
inhibit 5-LOX and COX-1 (a key enzyme that catalyzes the biosynthesis of prostaglandin), the
1
5-19
enzymes associated with various inflammatory diseases and cancer.
Coumaperine, a piperine
20
type amide alkaloid found in white pepper (Piper nigrum Linn.), structurally similar to
piperine, and functionalized with biologically active sites like, phenolic moiety, Michael
acceptor group and amide functionality. Literature evidences show that aforementioned
1
1,21-24
functionalities are beneficial for 5-LOX inhibition.
Hence, we thought it is worth

5
investigating coumaperine derivatives for 5-LOX inhibition. Moreover, coumaperine and its
2
5-27
derivatives are scarcely explored for biological applications.
In the present study we have
synthesized twenty four coumaperine derivatives and evaluated their 5-LOX inhibition in vitro.
For the active compounds, antioxidant, pseudoperoxidase activity and molecular docking studies
were carried out to understand their inhibitory mode of action. Molecular docking to the active
site of 5-LOX has been conducted and the results are compared with zileuton.
Scheme 1: Structure of coumaperine, piperine and selected 5-LOX inhibitors.
2
. Results and Discussion
2
.1. Chemistry
Piperine, an important constituent of black and long pepper is shown to inhibit enzymes such as,
COX-1 and 5-LOX (enzymes involved in biosynthesis of prostaglandins and leukotrienes
1
9
respectively). These enzymes are associated with various inflammatory diseases. It is
anticipated that coumaperine, a piperine type amide alkaloid present in white pepper with its
2
5
structure similar to piperine should also possess 5-LOX inhibitory activity. The concentration
of coumaperine in white pepper (Piper nigrum Linn.) is very minimal (6 ppm) and synthetic

6
methods known for the preparation of coumaperine and its derivatives suffer from limitations
2
8-31
such as more number of synthetic steps, expensive reagents, or tedious synthetic procedure.
This hampered the development of coumaperine derivatives for biological application. A simple
and robust synthetic method is highly desirable. Herein, we have adopted a simple and a
3
2
modified literature procedure for preparing coumaperine derivatives (Scheme 2). Twelve new
coumaperine derivatives (Scheme 3b) were synthesized and evaluated for 5-LOX inhibition.
Prior to this, we have evaluated 5-LOX inhibitory activity of other coumaperine derivatives that
2
7
we had previously analyzed for NF-kB inhibition (Scheme 3a).
Coumaperine derivatives were synthesized as outlined in the scheme 2. N-Crotonyl piperidine
or sorbyl piperidine) was reacted with variously substituted benzaldehydes in presence of a
(
base, KOH/t-BuOK (detail synthetic procedure is given in Experimental section). Coumaperine
was synthesized starting from crotonic acid. Crotonic acid was converted to crotonyl chloride
and reacted with piperidine to obtain N-crotonyl piperidine (Scheme 2). It was reacted with
tetrahydropyran protected 4-hydroxy benzaldehyde in presence of potassium tert-butoxide to
obtain tetrahydropyran protected coumaperine. Deprotection with TFA yielded coumaperine
with 24% overall yield. Following the same synthetic procedure the other coumaperine
derivatives, CP-9, 10, 27, 32, 38, 50, 102, 123, 146, 147, 154, 155, 184, 193 and 194 were
synthesized in excellent to moderate yield (99-12%). Acetylation of coumaperine (with acetic
anhydride) yielded acetyl coumaperine, CP-158 with moderate yield, 36%. The dihydroxyl
derivative, CP-209 was synthesized from piperine. Deprotection with BBr at -15 °C yielded
3
CP-209 with 44% yield. Initially, we attempted to prepare it from dimethoxy derivative, CP-10
using AlCl Only mono demethylated compound, CP-205 was formed as sole product. When
3
.
BBr was used, a mixture of mono and di demethylated product was formed. Mono demethylated
3

7
product (CP-205) was formed as a major product. CP-215 was synthesized from 3,4-
methylenedioxy cinnamic acid and piperidine. Knoevenagel condensation of 3,4-methylenedioxy
benzaldehyde with malonic acid yielded 3,4-methylenedioxy cinnamic acid. It was converted to
acid chloride and reacted with piperidine to obtain CP-215 with an overall yield of 39%.
Deprotection of CP-215 with Lewis acid (AlCl ) yielded CP-237 in moderate yield, 38%. The
3
trihydroxyl derivative, CP-262-F2 was synthesized from trimethoxy derivative, CP-27 (Scheme
4
). Demethylation with Lewis acid, BBr at -78 °C yielded mono demethylated product, CP-262-
3
F1 and tri demethylated product, CP-262-F2 with 33% and 14% yield respectively. The mono
demethylated product (CP-262-F1) can be further demethylated (BBr at -78 °C) to get the tri
3
demethylated product in 28% yield (Scheme 4). Efforts to isolate the di demethylated product
were not successful. Our initial efforts to deprotect CP-27 with LiCl, AlCl at room temperature,
3
pyridinium hydrochloride at 0 °C and BBr at 0 °C, resulted in black non-traceable material.
3
Demethylation with AlCl at 0 °C resulted solely in mono demethylated product (CP-262-F1,
3
Scheme 4).
Attempt to demethylate 2,4,6-trimethoxy derivative, CP-194 (2,4,6-trimethoxy derivative) to
obtain the corresponding trihydroxyl compound using Lewis acids, AlCl and BBr with varied
3
3
ratio of substrate to reagent at different temperature (30 °C, 0 °C, -20 °C, -78 °C), mode of
addition and different solvents were not fruitful.
All the synthesized coumaperine derivatives were characterized by various spectroscopic
1
13
techniques including, FT-IR, H NMR, C NMR and GC-MS. The previously unreported
coumaperine derivatives were additionally characterized by HRMS or elemental analysis. The
chemical structure of coumaperine derivatives, CP-155, CP-208 and CP-262-F2 (the
compounds that shwoed best 5-LOX inhibition) were unambiguously established by single

8
crystal X-ray diffraction (ORTEP diagram given in ESI). FT-IR spectrum of all the coumaperine
-1
derivatives showed a peak corresponding to amide functionality around 1627-1659 cm . The
-1
hydroxyl derivatives showed characteristic hydroxyl peak(s) around 3080-3514 cm . The
calculated molecular mass of all the derivatives matched with experimental data (GC-
1
13
MS/HRMS). H NMR and C NMR spectra matched with the structural features of the
derivatives. The coupling constant of alkene bonds were calculated to be 15-16 Hz, indicating
that alkene bonds are trans. The hydroxyl protons of CP, CP-205, 209, 237, 239, 262-F1, 262-F2
appeared between δ 8.5-9.95 ppm. The methylene protons of CP-155, 215 appeared between δ
5
.9-6 ppm. Piperidine protons appears as three distinct multiplets in the aliphatic region, δ1.4-3.5
ppm. All the aromatic and alkene protons appeared between δ6.1-7.6 ppm.
Scheme 2: General reaction scheme for synthesis of coumaperine derivatives.

9
Scheme 3: The coumaperine derivatives synthesized and evaluated for 5-LOX inhibition; a)
coumaperine derivatives that we previously reported for NF-kB inhibition; b) The newly
synthesized coumaperine derivatives for 5-LOX inhibition

1
0
Scheme 4: Synthesis of CP-262-F2 and CP-262-F1 from CP-27
2
.2. 5-LOX enzyme inhibition and its structure activity relationship (SAR) analysis
2
7
The coumaperine derivatives that were previously reported for NF-kB inhibition was screened
for 5-LOX inhibition (Scheme 3a). Based on these results, we rationally designed and
synthesized other coumaperine derivatives (Scheme 3b). Inhibition study was carried out at
fixed concentration of 20 µM. Inhibition potential of coumaperine derivatives was compared
with standard 5-LOX inhibitor, zileuton (Table 1, entry 14).
The initial screening study showed that the parent compound, coumaperine (CP) has moderate
inhibitory activity, 44.44% (Table 1 entry 9). The derivatives substituted with, -N(CH ) , (CP-
3
2
3
2, 13.4%), -OCyclopentyl (CP-184, 16.1%), -OAc (CP-158, 18.3%), -H (CP-9, 36.3%), -OCH3
CP-38, 42.6%) showed inhibitory activity inferior to coumaperine (Table 1, compare entry 9
with 2, 3, 4, 7, 8 respectively). Similarly, the inhibitory activity of CP-102 (1.1%) and 146
27.4%) were less than coumaperine (Table 1, entry 1 and 5). The 2,3,4- trimethoxy derivative,
(
(
CP-50 is also less potent (36.0%) than coumaperine. Whereas the 3,4,5-trimethoxy derivative,
CP-27 exhibited excellent activity (66.7%, Table 1, entry 12). The methylenedioxy (PIP,
4
7.5%) and dimethoxy (CP-10, 51.9%) derivatives also showed slightly better activity than

1
1
coumaperine (Table 1, entry 10 and 11). The alkene homologue of methylenedioxy derivative,
CP-155 (Table 1, entry 13, CP-155, a natural product, Piperettin, found in black pepper)
displayed superior inhibitory activity (76.0%) among the coumaperine derivatives evaluated in
the initial screening study.
The above results indicate that substituting hydroxyl group of coumaperine (CP) with electron
donating groups such as, –N(CH ) , –OCH electron withdrawing group such as, -OAc and
3
2
3,
unsubstitution resulted in derivatives of inferior activity (compare entry 9 in Table 1 with 1-8).
Whereas, substitution with methylenedioxy and multiple methoxy (di and tri) groups increase the
activity (compare entry 9 in Table 1 with 10-13). Although, substitution with multiple methoxy
group is beneficial, substitution pattern influences the activity (compare inhibitory activity of
CP-50 and 27 in table 1, entry 6 and 12). Increasing the alkene conjugation (di to tri) to
methylenedioxy derivative also increases the activity (Table 1, entry 10 and 13).
Table 1: 5-LOX inhibitory activity of coumaperine derivatives screened initially. Data are
expressed as means ± S.D of single determinations obtained in three independent biological
experiments.
%
5-LOX
S.No Compound
inhibition± SD
1
2
.
.
1.08±0.01
13.44±2.28
CP-32

1
2
3
4
.
.
16.13±1.52
18.28±4.56
CP-184
CP-158
5
6
.
.
27.42±0.76
36.02±8.36
CP-146
CP-50
7
8
.
.
36.27±1.46
42.59±1.80
CP-9
CP-38
CP
9
1
.
44.44±2.19
47.45±8.05
0.
PIP

1
3
1
1
1.
2.
51.85±3.10
66.67±2.00
CP-10
CP-27
1
1
3.
4.
76.02±1.46
90.52±1.60
CP-155
Zileuton
Since the trimethoxy substitution pattern influences the inhibitory activity, we have synthesized
the other trimethoxy derivative, CP-194 (2,4,6-trimethoxy) and dimethoxy derivative, CP-193
(
3,5-dimethoxy) and evaluated their 5-LOX inhibition. To study the influence of alkene
conjugation to methyelendioxy derivative, we synthesized the mono conjugated derivative, CP-
15 and compared its activity with its congeners PIP (di) to CP-155 (tri). Referring to the
literature we found that several catechol type compounds are potent 5-LOX inhibitors (Scheme
2
2
3,33
5
).
With this in mind we have synthesized the dihydroxyl derivatives, CP-209, 215 and 239.
Also, we have synthesized other coumaperine derivatives (CP-154, 123, 147) substituted with
halogens (–F, -Cl) and thiomethyl (–SCH ). The above synthesized coumaperine derivatives
3
were subjected for 5-LOX inhibition. The inhibitory activity was performed at fixed
concentration, 20 µM.

1
4
Scheme 5: Catechol type 5-LOX inhibitors reported in literature.
The 3,5-dimethoxy (CP-193) and 2,4,6-trimethoxy (CP-194) derivatives showed superior
activity (64.9%, 77.1% respectively, Table 2, entry 8 and 9) than their congeners, CP-10
(51.9%) and CP-27 (66.7%) respectively. This indicates, the substitution position of di and
trimethoxy indeed influence the 5-LOX inhibitory activity. As expected the mono conjugated
methylenedioxy derivative, CP-215 showed inferior activity (39.9%, Table 2, entry 4) than their
homologues, PIP and CP-155. This result further confirms that increasing the conjugation to
methylenedioxy derivative increases the 5-LOX inhibitory activity. The coumaperine derivatives
substituted with –SCH and –Cl groups were less potent (11.8% and 14.5% respectively, Table
3
2
, entry 1 and 2) than the parent compound, CP. Whereas, the –F substituted derivatives showed
slightly higher activity (48.7%, Table 2, entry 5) than parent compound, CP. Presuming that
increasing the number of fluorine group would increase the activity, we sought to prepare 3,4-
difluoro derivative. Attempts to prepare 3,4-difluoro derivative was not successful. To our
excitement the catechol type dihydroxyl derivative, CP-209 showed superior activity (82.8%,
Table 2, entry 10) among the coumaperine derivatives tested. Its 5-LOX inhibitory potential is

1
5
comparable to zileuton (90.5%), the only standard drug available in the market for 5-LOX
inhibition. This drug also poses severe side effects. Whereas, CP-209 a methylene deprotected
piperine, a natural product found in black pepper, represents that it could be developed as a
promising 5-LOX inhibitor. To our surprise, the corresponding mono and tri conjugated
dihydroxyl derivatives, CP-237 and 239 are less active (55.2% and 30.3% respectively, Table 2,
entry 6 and 3) than CP-209.
Table 2: 5-LOX inhibitory activity of second set of coumaperine derivatives. Data are expressed
as means ± S.D of single determinations obtained in three independent biological experiments.
%
5-LOX
S.No Compound
inhibition± SD
1
2
.
.
11.83±1.52
CP-147
CP-123
14.52±2.28
3
4
.
.
30.34±1.18
39.88±4.21
CP-239
CP-215

1
6
5
6
.
.
48.70±2.92
55.17±4.71
CP-148
CP-237
CP-205
7
8
9
1
.
55.49±4.21
64.89±4.21
77.12±1.68
82.75±3.25
.
CP-193
CP-194
CP-209
.
0.
With an aim to increase the activity further, we decided to increase the number of hydroxyl
groups to CP-209. Hence, the trihydroxyl derivative, CP-262-F2 was synthesized (Scheme 3b)
and assessed for 5-LOX inhibition.
To our disappointment the trihydroxyl derivative CP-262-F2 showed slightly lower inhibitory
activity (82.5%, Table 3 entry 2) than CP-209 (82.8%, Table 2 entry 10). In the process of
preparation of trihydroxyl derivative we obtained the monohydroxy derivative, CP-262-F1. As
expected, CP-262-F1 showed lower activity (32.6%, Table 3 entry 1) than CP-262-F2.

1
7
The IC value was determined for the best active compounds, CP-209 and CP-262-F2. Their
5
0
IC values 2.1 and 2.3 µM respectively are comparable to prominent 5-LOX inhibitor, zileuton,
5
0
IC = 1.4 µM and superior to other known, 5-LOX inhibitors such as, pterostilbene, (IC =
5
0
50
9
4
.32±3.32 µM, zileuton = 4.23±3.00 µM), oxyresveratrol (IC = 18.49±7.79 µM, zileuton =
50
2
2
.23±3.00 µM), idebenone (>10 µM, zileuton = 0.59±0.1).
In essence, the above structure activity relationship demonstrates that the coumaperine
derivatives substituted with multiple (di and tri) hydroxyl and methoxy groups are beneficial for
5
-LOX inhibition.
Table 3: 5-LOX inhibitory activity of third set of coumaperine derivatives. Data are expressed as
means ± S.D of single determinations obtained in three independent biological experiments.
%
5-LOX
S.No Compound
inhibition± SD
1
.
.
32.66±0.71
CP-262-F1
CP-262-F2
2
82.47±2.48
2
.3. Antioxidant studies
Numerous LOX inhibitors are known to exhibit inhibitory activity via antioxidant
3
4,35
mechanism,
by either preventing the oxidation of iron at the active site or by scavenging the
2
radicals at the active site and thereby inhibiting the enzyme activity. It is well established that

1
8
the redox activity of iron at the active site plays crucial role in biosynthesis of pro-inflammatory
2
mediators, leukotrienes and lipotoxins. Thus, evaluation of antioxidant potential of LOX
inhibitors would unveil whether their mode of inhibition is through redox mechanism or through
other pathways (iron-ligand chelators, non-redox competitive inhibitors or allosteric inhibitors).
Therefore, evaluation of antioxidant potential becomes an essential part of LOX inhibition study.
Antioxidant potential of coumaperine derivatives such as, CP-155, CP-194, CP-209 and CP-
2
62-F2 has been evaluated by DPPH assay. Results are shown in table 4.
Table 4: 5-LOX inhibitory activity and antioxidant activity of the highly active coumaperine
derivatives (at 20 µM). Data are expressed as means ± S.D of single determinations obtained in
three independent biological experiments.
S.
No.
%5-LOX
inhibition ± SD
% Scavenging
of DPPH ± SD
Compound
1
.
82.75 ± 3.25
82.47 ± 2.48
76.76 ± 0.25
71.33 ± 1.25
CP-209
2
.
CP-262-F2
3
4
.
.
76.02 ± 1.46
77.12 ± 1.68
0.90 ± 0.51
3.60 ± 1.78
CP-155
CP-194

1
9
5
.
-
80.1 ± 2.15
Ascorbic acid
CP-209 a catechol type dihydroxyl derivative and CP-262-F2 a vicinal trihydroxyl derivative
effectively scavenged the DPPH radicals. They scavenged 76.7% and 71.3% of DPPH radicals
respectively (measured by DPPH assay). This study indicates that CP-209 and CP-262-F2
excellent antioxidants and their mode of action is via redox process. They may either scavenge
the radicals generated at the active site or involve in a combination of iron ion-chelation and iron
ion reduction. To further confirm that these inhibitors follow redox inhibitory pathway,
pseudoperoxidase assay was conducted (Section 2.4).
On the other hand, CP-155 and CP-194 showed poor antioxidant activity, they scavenged only
0
.9% and 3.6% of DPPH radicals respectively (measured by DPPH assay). This shows that CP-
55 and CP-194 are unlikely to follow redox pathway.
1
2
,10
Literature references
show that 5-LOX inhibitors lacking iron ion-chelating functional groups
such as, hydroxymic acid, N-hydroxyl urea or hydroxyl functionality are unlikely to inhibit
through 5-LOX via iron ion-chelation pathway. Since, CP-155 and CP-194 lack aforementioned
functional groups, they might follow other inhibitory pathways such as, non-redox competitive
or allosteric inhibition. To explore this, CP-155 and CP-194 were docked to the active site of 5-
LOX.

2
0
2
.4. Pseudoperoxidase activity for redox mechanism
It is well established that few compounds disrupt the redox cycle of the iron present in the
enzyme thereby inhibiting its catalytic activity. Compounds with high antioxidant activity may
be doing this. To analyze this particular mechanism, a specific 5-LOX assay for some of the best
3
6,37,38
coumaperine derivatives have been evaluated through pseudoperoxidase activity assay.
In
the presence of redox inhibitor, ferrous ion of 5-LOX consumes lipid peroxide, 13(S)-
hydroperoxyoctadecadienoic acid (13(S) HpODE) which can be quantified by the decrease in
absorbance at 234 nm and thus their pseudoperoxidase activity can be determined. This activity
has been determined for the inhibitors CP-209, CP-262-F2, CP-155 and CP-194 along with the
reference, Zileuton (Figure 1). It has been found that in presence of both the inhibitors, CP-209
and CP-262-F2, absorbance decreased by 57.4 % and 50.8 % (Figure 1b & c) respectively in
3
00 seconds as similar to the reported redox standard (42.1%) indicating that there is
considerable degradation of hydroperoxide product. This data strongly indicates that both
compounds follow redox mechanism. Whereas for compounds CP-155 and CP-194, the
absorption remained constant throughout the process (Figure 1d & e) indicating that there is no
change in the redox process or any change in the oxidation state of iron ion involved for the
inhibition of enzyme, hence their mechanism of action is via non-redox pathway.

2
1
Figure 1: 5-LOX pseudoperoxidase activity assay plot for redox mechanism at 234 nm (absorbance
subtracted from control) for the consumption of substrate 13(S)-HpODE by a) Zileuton, b) CP-209 c)
CP-262-F2 and d) CP-155 and e) CP-194

2
2
Scheme 6: Schematic representation of pseudoperoxidase assay
2
.5. Molecular docking studies: Active Site Docking
To support the observed 5-LOX inhibitory activity of CP-155 and 194, they were docked to
active site of 5-LOX enzyme (PDB code: 3O8Y), binding energy was calculated and the results
were compared with zileuton and arachidonic acid (AA).
Docking, CP-155 and 194 to the active site showed that they were stabilized by vander Waals
interaction formed with the residues Phe177, Tyr181, Phe359, Gln363, Leu368, His372, Ile406,
Asn407, Ala410, Leu414, Ile415, Phe421, Asn425, His600, Ala603, Val604, Leu607 and Fe at
the active sites (Figure 2a and 2b). Both the inhibitors, CP-155 and 194 exhibited strong
binding affinity (-3.19 kcal/mol and -2.64 kcal/mol respectively) than zileuton (-2.61 kcal/mol,
Table 5). Zileuton was stabilized by hydrogen bond interactions (Figure 2c) at the active site.
The N-hydroxyl proton of the zileuton forms hydrogen bond with oxygen of amino acid Gln363
and the nitrogen atom of imidazole ring of His367. Though CP-155 and 194 favoured similar

2
3
hydrophobic interactions as arachidonic acid, in addition the oxygen atom of arachidonic acid
formed hydrogen bond interactions with the residues Asn425 and His600 (Figure 2d).
Arachidonic acid exhibited higher binding score (-5.86 kcal/mol) than both CP-155 and 194.
Therefore, it is unlikely that CP-155 and 194 can compete with arachidonic acid for the active
site. Suggesting that CP-155 and CP-194 are non-competitive inhibitors.
Table 5: The binding energy (∆G ), intermolecular energy (∆Gintermol), internal energy
BE
(∆Ginternal) and torsional energy (∆Ginternal) of CP-155, CP-194, AA, zileuton with 5-LOX are
given below. All the energies are reported in kcal/mol. The compounds were docked into the
active site cavity of 5-LOX enzyme (PDB ID: 3O8Y).
∆
∆
Gintermol
Ligands
∆GBE
∆Ginternal
∆Gtor
Gvdw_hb_desol
∆Gelec
-0.03
-0.17
0.82
CP-155
CP-194
Zileuton
AA
-3.19
-2.64
-2.61
-5.86
-4.35
-3.84
-4.33
-7.40
-0.48
-0.85
0.24
1.19
1.79
0.89
2.98
-1.45
-0.19
*AA represents Arachidonic Acid

2
4
(a)
(b)

2
5
(c)
(d)

2
6
Figure 2: Docked conformation of the compounds (a) CP-155 (b) CP-194 (c) arachidonic acid
and (d) protonated zileuton, with crystal structure of 5-LOX (PDB code: 3O8Y). The compounds
were docked into the active site cavity of 5-LOX enzyme and the compounds are represented in
stick model.
2
.6. In-Silico ADME/TOX analysis
ADME/TOX corresponds to Adsorption, Distribution, Metabolism and Excretion /toxicity
properties of drug candidates.
Nearly 40% of drug candidates undergoing clinical trials fail due to their poor ADME or toxicity
properties. We did ADME/TOX analysis to understand the synthesized compound’s drug
likeliness and toxicity to avoid future failure of drug testing in vivo, thereby streamlining the
overall development process.
For the active compounds, CP-155, 194, 209 and 262-F2 ADME/TOX study was conducted.
The computational ADME/TOX analysis of CP-155, 194, 209 and 262-F2 for 14 molecular
descriptors were computed using QikProp(Table 6). Additional set of 12 descriptor were
computed using ADMETSAR (http://lmmd.ecust.edu.cn/admetsar2) (Table 7). Most of the
molecular descriptors such as molecular weight, CNS Activity, QLogBB, QlogKhsa, QlogKp
were within the range of 95% of present approved drugs. In contrast CP-194, CP-209, CP-262-
F2 have QPLoghERG values showing slight negative deviation from the recommended range.
On the front of biological evaluation for toxicity descriptors such as ames mutagenesis,
carcinogenicity, eye corrosion predicted “negative”. All the compounds show reduced
hepatotoxic probability score than zileuton (standard 5-LOX inhibitor), suggesting that the
synthesized compounds are safe for biological administration and can be potent anti-
inflammatory drugs. The evaluation for hepatic site of metabolism predict that the metabolism of

2
7
CP-155 occurs at CYP1A2, CP-194 and CP-209 are both metabolize by CYP2C9, and CP-262-
F2 is metabolized by CYP1A2, CYP2D6 and CYP34A.
Table 6: Computational Analysis of ADME for Coumaperine Derivatives using QikProp 5.5
Molecule
CP-155
CP-194
CP-209
CP-262-F2 Range of 95% of Drugs
-
+
2 (inactive) to
2 (active)
CNS Activity
Human Oral
0
-2
-2
0
3
3
3
3
1 - 3
A
Absorption
Mol.Wt
331.411
273.331
289.33
311.38
130 – 725.0
<
25% is Poor
>80% is High
± 20 percent)
%
Oral
1
00
88.624
76.58
100
Absorption
(
QPlogBB
-0.345
-5.00
0.203
-1.13
4.06
-1.268
-5.21
0.016
-2.884
2.468
-3.869
435.37
201.372
0
-1.81
-5.47
-0.155
-3.782
1.776
-3.574
155.659
66.25
0
-0.273
-5.10
0.191
-0.88
3.969
-4.363
3996.478
2211.526
0
-3 to 1.2
QPloghERG
QPlogKhsa
QPlogKp
Concern Below -5
-1.5 to 1.5
-8 to -1.0
QPlogPo/w
QPlogS
-2 to 6.5
-4.413
3876.095
2139.61
0
-6.5 to 0.5
QPPCaco
QPPMDCK
Rule Of Five
<25 Poor; > 500 Great
<25 Poor; > 500 Great
Maximum 4
B
C
Rule Of Three
0
0
0
0
Maximum 3
A
B
denotes predicted quantitative human oral absorption 1, 2 or 3 for low, medium and high respectively; Denotes Number of
Violation of Lipinski’s rule, the rules are mol_wt <500, QpLog Po/w < 5 donorHB <5, accptHB ≤ 10. Compound satisfying these
C
rules are considered drug-like; Denotes number of violation of Jorgensen rule. The rules are QPlogS > -5.7, QPPCaco > 22
nm/s, # Primary Metabolites < 7. Compounds with less number of violation of these rules are more likely to be orally available.
K
TABLE 7: Computational analysis for Toxicity and Metabolism prediction using AdmetSAR
Molecule
CP-209
CP-194
CP-155 CP-262-F2 Zileuton
Ames mutagenesis
Carcinogenicity
-
-
-
-
-
-
-
-
-
-
+
+
+
+
Hepatotoxicity
(probability: (probability: -
(probability: (probability:
0
.525)
0.575)
0.525)
0.875)
Human either-a-go-
go inhibition
Eye corrosion
-
+
-
-
-
-
-
-
-
-

2
8
CYP1A2 inhibition
CYP2C19 inhibition
CYP2C9 inhibition
CYP2C9 substrate
CYP2D6 inhibition
CYP2D6 substrate
CYP3A4 inhibition
CYP3A4 substrate
-
-
-
+
-
-
-
-
-
-
-
+
-
-
-
-
+
-
-
-
-
-
-
-
+
-
+
-
-
-
-
+
-
+
-
-
+
-
-
-
‘
-’ Denotes Negative; ‘+’ Denotes Positive. Cytochrome P450 (CYP450) substrate, inhibitor - Substrate:
CYP2C9, 2D6, 3A4. Inhibitor: CYP1A2, 2D6, 2C9, 2C19, 3A4.
For the active compounds, CP-155, 194, 209 and 262-F2, MouseTox was also performed to
predict the cytotoxic effect to NIH/3T3 (mouse embryonic fibroblast) cells through Enalos Cloud
Platform (http://enalos.insilicotox.com/MouseTox). All the compounds were found to be
inactive. Suggesting that the compounds are non-cytotoxic to NIH/3T3s.
TABLE 8: In Silico prediction of cytotoxic effect to NIH/3T3 cells using MouseTox
Compound Prediction (class)
CP155
CP194
CP209
CP262F2
Inactive
Inactive
Inactive
Inactive
“
Active” (compounds cytotoxic to NIH/3T3s) and “inactive” (compounds non-cytotoxic to NIH/3T3s)
2
.7. Insight into mechanism of action of CP-155, 194, 209 and 262-F2
Based on the mode of inhibition, 5-LOX inhibitors are classified as (a) redox inhibitors
scavenge lipidperoxides and reduce iron at active site. Several phenolic compounds claimed to
(
be redox inhibitors ex. NDGA, cirsiliol etc.), (b) iron ligand inhibitors (chelate the active site
iron, ex. zileuton, atreleuton), (c) non-redox competitive inhibitors (compete with AA or lipid
peroxides, ex. setileuton, L-697198) and (d) allosteric inhibitors (bind to non-active site of the
enzyme ex. licofelone and AKBA) (Scheme 7). The inhibitors, atreleuton, setileuton, licofelone,
CJ-13610 are under clinical trial and zileuton is the only approved drug marketed that targets 5-

2
9
LOX. It is believed to inhibit 5-LOX via both iron ion-chelation and reduction of ferric
2
,4,10,33,39,40
iron.
In the present study, CP-209 a catechol type dihydroxyl compound (a methylene deprotected
piperine. Piperine, an active ingredient of black pepper extensively investigated for various
biological applications) and CP-262-F2 a vicinal trihydroxyl compound displayed highest 5-
LOX inhibitory activity, ~83% each. At the same time both the compounds effectively
scavenged DPPH radicals (76.7% and 71.3% respectively) and decreased the absorbance by
>50% in pseudoperoxidase assay. Taken together the high 5-LOX inhibition, excellent
antioxidant activity and considerably degradation of 13(S)-HpODE in pseudoperoxidase assay,
strongly indicates that both compounds follow redox pathway (Scheme 7). On the other hand
both CP-155 (Piperettin, a natural product found in black pepper) and 194 a 2,4,6-trimethoxy
derivative poorly scavenged DPPH radical and no notable degradation of hydroperoxide in
pseudoperoxidase assay. Suggesting that their mode of inhibition is not through redox pathway.
Molecular docking of CP-155 and 194 to the active site of 5-LOX (PDB: 3O8Y) and binding
energy calculation with reference to zileuton (standard 5-LOX inhibitor) and arachidonic acid
indicates that CP-155 and 194 are non-competitive inhibitors.