Synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-LOX) inhibition activities, and molecular docking study of 7-substituted coumarin derivatives

Article abstract of DOI:10.1016/j.bioorg.2016.06.004

In the present study, 7-subsituted coumarin derivatives were synthesized using various aromatic and heterocyclic amines, and evaluated in vivo for anti-inflammatory and analgesic activity, and for ulcerogenic risk. The most active compounds were evaluated in vitro for 5-lipoxygenase (5-LOX) inhibition. Docking study was performed to predict the binding affinity, and orientation at the active site of the enzyme. In vivo anti-inflammatory and analgesic activity, and in vitro 5-LOX enzyme inhibition study revealed that compound 33 and 35 are the most potent compounds in all the screening methods. In vitro kinetic study of 35 showed mixed or non-competitive type of inhibition with 5-LOX enzyme. Presence of [Formula presented]₃ group in 35 and [Formula presented] in 33 at C6-position of benzothiazole ring were found very important substitutions for potent activity.

Full text of DOI:10.1016/j.bioorg.2016.06.004

Accepted Manuscript
Synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-LOX) inhibition ac-
tivities, and molecular docking study of 7-substituted coumarin derivatives
Pavan Srivastava, Vivek K. Vyas, Bhavesh Variya, Palak Patel, Gulamnizami
Qureshi, Manjunath Ghate
PII:
S0045-2068(16)30083-9
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.06.004
YBIOO 1918
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
3 May 2016
16 June 2016
17 June 2016
Please cite this article as: P. Srivastava, V.K. Vyas, B. Variya, P. Patel, G. Qureshi, M. Ghate, Synthesis, anti-
inflammatory, analgesic, 5-lipoxygenase (5-LOX) inhibition activities, and molecular docking study of 7-substituted
coumarin derivatives, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.06.004
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Synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-LOX)
inhibition activities, and molecular docking study of 7-substituted
coumarin derivatives
Pavan Srivastavaa, Vivek K. Vyasa, BhaveshVariyab, Palak Patelc, Gulamnizami Qureshia,
Manjunath Ghatea*
aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University,
Ahmedabad 382481, Gujarat, India
bDepartment of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad 382481,
Gujarat, India
cInstitute of Science, Nirma University, Ahmedabad 382481, Gujarat, India
Abstract
In the present study, 7-subsituted coumarin derivatives were synthesized using various aromatic
and heterocyclic amines, and evaluated in vivo for anti-inflammatory and analgesic activity, and
for ulcerogenic risk. The most active compounds were evaluated in vitro for 5-lipoxygenase (5-
LOX) inhibition. Docking study was performed to predict the binding affinity, and orientation at
the active site of the enzyme. In vivo anti-inflammatory and analgesic activity, and in vitro 5-
LOX enzyme inhibition study revealed that compound 33 and 35 are the most potent compounds
in all the screening methods. In vitro kinetic study of 35 showed mixed or non-competitive type
of inhibition with 5-LOX enzyme. Presence of -OCH3 group in 35 and -Cl in 33 at C6-position
of benzothiazole ring were found very important substitutions for potent activity.
Key words: 7-Substituted coumarins; Anti-inflammatory and analgesic agents; 5-Lipoxygenase
inhibitors; Docking
*corresponding author. (M.D. Ghate)
Telephone number +91 9879609651
Email address- ghate72@gmail.com
1

1. Introduction
Inflammation is a multifactorial dynamic physiological process that occurs in response to
infection or injury in the body in an attempt to eliminate or limit the spread of the injurious
material or agent, and promote tissue repair [1]. It is mediated by the sequential release of
mediators including; bradykinin, cytokines, vasoactive amine, eicosanoids, chemokines,
interleukins and growth factors. Symptoms of inflammation are pain, heat, swelling, edema and
redness [2]. Cyclooxygenases (COXs), lipoxygenases (LOXs), nitric oxide synthases (NOS) are
the key enzymes in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins, which
play a crucial role in stimulating inflammatory reactions. Inflammation is hallmark of many
diseases and linked to stroke, cancer, cardiovascular and neurodegenerative diseases [3].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs for the
treatment and management of inflammation. NSAIDs suppress the production of prostaglandins
(PGs), which is a main precursor for the biosynthesis of many inflammatory mediators, which
stimulates inflammatory reactions in the body [4]. These drugs block the arachidonic acid
metabolism via inhibition of several enzymes involved in the synthesis of PGs. Main side effects
associated with NSAIDs are gastrointestinal hemorrhage, kidney failure and ulceration, due to
changes in the physiological balance of PGs [5]. Because of these side effects with existing
NSAIDs, there is a need of novel agents with better therapeutic profile as anti-inflammatory
drugs. Leukotrienes (LTs) are one of the mediators of inflammation, which are derived from the
oxidation of polyunsaturated fatty acids in the presence of 5-lipoxygenase (5-LOX) enzyme. LTs
are mediators of many disorders related with inflammatory processes via the interaction with the
LTB4, BLT1 and BLT2 receptors. Lipoxygenases (LOXs) are a member of family of non-heme
iron-containing dioxygenases, which are responsible for the formation of hydroperoxy
metabolites (HPETEs) by the addition of molecular oxygen to the substrate, and are found in
animals, plants and fungi. In humans six functional isoform of LOXs are present. Inhibitors bind
with active site of lipoxygenase and exhibit competitive type of inhibition. LTs inhibitors are
divided in three category 1) redox-active compounds 2) iron-ligand inhibitors with weak redox-
active properties, and 3) non-redox-type inhibitors [6]. Coumarin derivatives are redox-active
inhibitors of LOXs [7]. Coumarin consists of a benzene ring attached to a pyrone ring and
classified as a member of the benzopyrone family of compounds. Coumarin is the parent
compound of the coumarin family, which is the large class of naturally occurring phenolic
2

compound [8]. In this work, we designed and synthesized 7-substituted coumarin derivatives
using various aromatic, and heterocyclic amines. The synthesized compounds were characterized
1
by FTIR, H, 13C NMR, mass spectroscopy (MS) and elemental analysis. The synthesized
compounds were also docked with 5-LOX enzyme to predict the binding affinity, and orientation
at the active site of the enzyme. Synthesized compounds were tested in vivo for anti-
inflammatory and analgesic activity, and selected potent compounds were screened in vitro as 5-
LOX inhibitors.
2. Materials and methods
2.1. Chemistry
Digital melting point apparatus and open capillary methods were used for the determination of
melting points (mp) of intermediate and final compounds, which were found uncorrected.
Progress of chemical reactions were monitor by TLC using precoated silica plates. For detection
of TLC spots UV and iodine chambers were used. FTIR spectra were recorded on SHIMADZU
FTIR 6100 by KBr dispersion method. Mass spectra were recorded using ESI as ion source
1
(Agilent-6310 ion trap). H NMR spectra were recorded at room temperature on a Bruker AM-
13
400 and on Bruker Avance II 600 spectrometer, and C NMR spectra at 100 MHz in DMSO-d6.
Chemical shifts were measured in parts per million (ppm, δ) downfield from an internal
tetramethylsilane (TMS) standard. For the purification of compounds column chromatography
was used. Perkine Elmer elemental analyzer was used for the elemental analysis. Chemicals were
purchased from Spectrochem, Aldrich, Himedia, SD-Fine Merck and Sigma.
2.1.1 Synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one (3) (Scheme 1) [9]
In a 3-litre three-necked round bottom flask fitted with a thermometer, mechanical stirrer and a
dropping funnel, was charged with 1 liter of concentrated sulphuric acid in an ice bath.
Temperature of RBF falls below 100C, a solution of 100 g (0.91 mol) of resorcinol in 134 g
(130.5 ml, 1.03 mol) of redistilled ethyl acetoacetate was added drop wise with stirring.
Temperature was maintained below 100C by means of an ice-salt bath during the addition (2 h).
Reaction mixture was kept at room temperature for about 18 h, then poured with vigorous
stirring into a mixture of 2 kg of crushed ice and 3 liter of water. Precipitates were collected by
suction, filtered and washed with cold water (25 ml X 3). Solid residues were dissolved in 1500
3

ml of 10% sodium hydroxide solution, filtered and 2N sulphuric acid (550 ml) was added with
vigorous stirring until a solution became acid to litmus. Crude 4-methyl-7-hydroxycoumarin was
collected after filtration at the pump, washed with cold water (25ml X 4), and dried.
Recrystallized with 95% ethanol, the pure compound separated as colorless needles, in the yield
of 155 g (97%). MP was 185-1860C.
2.1.2 Synthesis of 4-methyl-7-(oxiran-2-ylmethoxy)-2H-chromen-2-one (5) [10]
7-Hydroxy-4-methyl-2H-chromen-2-one (3) (2.0 g, 8.85 mmol), and anhydrous K2CO3 (1.83 g,
13.3 mmol) were added in epichlorohydrin (4) (75 mL) and refluxed for 1 h. After completion of
the reaction, K2CO3 was filtered off and epichlorohydrin was removed in vacuum. The residue
was diluted with water and extracted with CHCl3 (75 mL X 3). The organic layer was washed
with water, brine, and dried over anhydrous Na2SO5. Column chromatography over silica gel
(100–200 mesh), and elution with 18% ethyl acetate in hexane furnished the epoxide (5).
Compound 5a was obtained as a side product.
2.1.3 Synthesis of 7-substituted-(2-hydroxy-3-(phenylamino)propoxy)-4-methyl-2H-
chromen-2-one derivatives(series 1) (7-28)
4-Methyl-7-(oxiran-2-ylmethoxy)-2H-chromen-2-one (5) (0.2 g, 0.70 mmol) and various
substituted aniline (6a-v) (1.06 mmol) were dissolved in ethanol (15 mL) and refluxed for 5-6 h.
After completion of the reaction excess ethanol was removed through high vacuum and the
residue was diluted with water and extracted with ethyl acetate. The organic layer was washed
with water, brine, and dried over anhydrous Na2SO4. Column chromatography over silica gel
(100–200 mesh) and elution with 50% chloroform in hexane furnished the target compounds (7-
28) of series 1.
2.1.4 Synthesis of substituted-2-aminobenzothizole derivatives (30a-e) [11]
A mixture of substituted anilines (29a-e) (0.01 mol) and potassium thiocyanate (28.84 g, 0.082
mol) were added to glacial acetic acid (20 ml), pre-cooled to 0°C in flat bottom flask. A solution
of bromine (17.35 g, 5.59 ml) in acetic acid (30ml) was added slowly with constant stirring to the
above mixture. Temperature was maintained at 0°C throughout the addition. Reaction mixture
was stirred at additional 2 h at 0°C and at room temperature for 10 h, and allowed to stand
overnight. Water (6 ml) was added, and the slurry was heated to 85°C on a steam bath and
4

filtered hot. The residue was placed in a reaction flask, and treated with glacial acetic acid (10
ml) heated again at 85°C and filtered hot. Filtrate was cooled and neutralized with concentrate
ammonia solution to pH 6. The precipitates were collected, recrystallized from 80% ethanol.
2.1.5 Synthesis of Substituted 7-(3-(benzo[d)thiazol-2-ylamino)-2-hydroxypropoxy)-4-
methyl-2H-chromen-2-one derivatives (31-35). (Series 2) (Scheme 2)
4-Methyl-7-(oxiran-2-ylmethoxy)-2H-chromen-2-one (5) (0.2 g, 0.70 mmol) and various
substituted 2-aminobenzothizole derivatives (30a-e) (1.06 mmol) were dissolved in ethanol (15
mL) and refluxed for 5-6 h. After completion of the reaction excess ethanol was removed
through high vacuum and the residue was diluted with water and extracted with ethyl acetate.
The organic layer was washed with water, brine, and dried over anhydrous Na2SO4. Column
chromatography over silica gel (100–200 mesh) and elution with 50% chloroform in hexane
furnished the target compounds (31-35) of series 2.
2.2 Molecular modeling study
2.2.1. Comparison of binding site of human 5-LOX with plant (soybean) 3-LOX
Lipoxygenases (LOXs) are found in animals, plants and fungi. Soybean lipoxygenase (linoleate
13S-lipoxygenase) is frequently used to screen compounds for LOX inhibition [7,12]. We also
performed in-vitro soybean LOX inhibition assay for screening of synthesized compounds as 5-
LOX inhibitors. Reddy et al. [13] compared binding sites of soybean lipoxygenase with human
5-LOX, and reported that both shared almost the same similarity with each other. We also
compared amino acid binding sites in human 5-LOX and soybean 3-LOX by the comparison of
physicochemical properties using MultiBind program at http://bioinfo3d.cs.tau.ac.il/MultiBind/.
First of all binding site residues of human 5-LOX (PDB ID: 3v99) [14], and soybean 3-LOX
(PDB ID: 1ik3) [15] for 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid were identified
using COFACTOR server at http://zhanglab.ccmb.med.umich.edu/COFACTOR/ [16], than these
protein structures (1ik3 and 3v99) as COFACTOR output were used as input for MultiBind
server.
5

2.2.2. Molecular docking study
Docking was carried out using Genetic Optimization for Ligand Docking (GOLD) 5.2.2 software
based on the GoldScore fitness function that uses the Genetic algorithm (GA), and considers full
ligand conformational flexibility, and partial protein flexibility. All water molecules, and hetero
atoms were omitted from the protein to evaluate the two scoring functions in GOLD. Docking
was performed with the crystal structures of 5-LOX (PDB ID: 3V99) [14], and all the
synthesized compounds. The binding site of the 5-LOX was known from the COFACTOR
server. A radius of 10 Å was used to direct site location. For each of the 10 independent GA
runs, a maximum number of 50000 GA functions were established. Automatic GA settings were
enabled by clicking on the Automatic radio button and the Search efficiency was set to 100%.
Rest of the settings was kept as default and the docking run was performed.
2.3 Pharmacological screening
2.3.1 Experimental animals
Adult, healthy, swiss albino mice of either sex having weight 25 to 30 g, and wistar rats of either
sex weighing from 250-300 g were utilized for the study, and experimental protocols carried out
in present study were permitted by the Institutional Animal Ethics Committee (IAEC) of Institute
of Pharmacy, Nirma University (vide protocol number: IPS/PCHEM/MPH11-12/2012). All the
experimental methods are in harmony with CPCSEA guidelines, Ministry of Environment and
Forests, Government of India. The animals were acclimatized in controlled laboratory
environment (12 h light/dark cycle, 20-22oC at 45-65% RH) and were allowed to access food and
water ad libitum.
2.3.2 Anti-inflammatory activity [17]
Synthesized compounds of both the series were evaluated for their anti-inflammatory activity
using carrageenan-induced rat paw oedema model. Paw oedema was induced by administration
of carrageenan into sub-planter region. Briefly, all the animals were randomly divided into 12
groups 5 animals in each, namely vehicle control (0.5% CMC, p.o.), standard treated (Diclofenac
sodium, 50 mg/kg, p.o.), and 10 groups were from test drug treatment (50 mg/kg in CMC, p.o.).
After overnight fasting, animals were pre-treated with the respective treatment and after 1h
treatment period, all the animals were intoxicated with carrageenan. 0.1 ml of 1% carrageenan in
6

normal saline was injected in sub-planter region of right hind limb of each animals and paw size
was measured in mL using Plethysmometer after 1 h, 2 h, 3 h and 6 h of the intoxication.
2.3.3 Analgesic activity
2.3.3.1 Acetic acid induced writhing method [18]
Mice were arbitrarily divided into 12 groups having 6 mice in each group, viz. normal saline
control (0.5% CMC solution), standard treated (Diclofenac sodium, 25 mg/kg, p.o.), and 10
groups were from test drug treatment (25 mg/kg in CMC, p.o.). Mice were overnight fasted and
pre-treated with aforementioned drugs. 30 min after the prior treatment, 0.25 ml of 0.6% v/v
solution of acetic acid was administered intraperitoneally. Animals were allowed to free
movement, and number of writhing were observed for 30 min. After the experimental procedure,
animals were rehabilited.
2.3.3.2 Radiant heat method (Tail-flick method) [19]
Synthesized compounds, which showed significant docking score, anti-inflammatory activity and
antinociceptive potential were further tested for their in vivo analgesic activity by Tail-flick
method with slight modification. All the rats were divided in 7 groups, 6 animals in each group,
viz. vehicle control (0.5% CMC, p.o.), standard control (Diclofenac sodium, 50 mg/kg, p.o.), and
6 groups were from test drug treatment (50 mg/kg in CMC, p.o.). Rats were closely restrained in
such way that the lower portion of the tail was immersed in warm water constantly maintained at
50oC. All animals were pre-treated with synthesized compounds before 1h of the experiment,
and the time at which rat flicks tail due to pain in hot water was recorded in seconds. The
reaction time was recorded at 60 min, 90 min and 120 min after the treatment, and increase in
reaction time was recorded. The maximum reaction time was restricted to 15 seconds to avoid
the damage due to heat.
2.3.4 Ulcerogenic potential [20]
The compounds of series 2 (31- 35) were further evaluated for ulcerogenic potential. All the
animals were randomized into 7 groups, 4 animals in each group, i.e. vehicle control (0.5%
CMC, p.o.), standard control (aspirin, 150 mg/kg, p.o.), and 6 groups were from test compounds
treatment (150 mg/kg in CMC, p.o.). Animals were exposed to test compounds for 7 subsequent
7

days, and after 24 h fasting, animals sacrificed with ether overdose. Incision was done on
peritoneal cavity, stomach was removed and place in saline plate. A longitudinal incision along
with greater curvature was made with the help of the sharp scissor, and presence and absence of
gastric irritation was observed macroscopically and lesions area were measured in term of mm2.
2.3.5 In-vitro LOX inhibition assay [21,22]
Synthesized compounds of series 2 (31-35) were found active in aforementioned protocol and
evaluated for LOX inhibitory activity. The in-vitro analysis for lipoxygenase inhibitory activity
was performed using Lipoxidase (LOX, linoleate: oxygen oxidoreductase, EC 1.13.11.12) from
Glycine max (soybean). It is determined by kinetic mode of spectro-photometric determination
method, which was performed by recording the rate of change in absorbance at 234 nm. It is
based on the principle that the increase of absorbance at 234 nm due to formation of 9- and 13-
hydroperoxides by LOX with substrate (linoleic acid). This reaction does not occur as the
enzyme gets inhibited due to addition of inhibitor in it. Briefly, the stock solution of
lipoxygenase was prepared by dissolving 10,000 units/ml of Lipoxidase in 200 mM borate
buffer, pH 9, and 5 µL of 80 mmol linoleic acid. Six different concentrations of inhibitors were
selected, and made for determining the enzyme inhibition activity. Increasing concentrations of
inhibitors were added to enzyme solution and kept for 5 min followed by substrate addition and
kept at room temperature with stirring for 20 min. One sample was also kept which does not
contain any inhibitor instead it contain vehicle of inhibitor solution. It account for 100%
enzymatic reaction. Blank contain all the substances except the enzyme solution to account for
non-enzymatic reaction. The reaction rates were compared, and the percentage inhibition due to
increasing concentration of inhibitors was calculated. The concentration of each test compound
was recorded in triplicate, and their IC50 values were determined graphically from absorption v/s
concentration curve.
2.3.6 In-vitro kinetic study
Continuous Spectro-photometric rate determination method was used to identify the type of
inhibition. Substrate (linoleic acid) was used in different concentrations (0.01 to 1 µM) prepared
in tween-20 in 200 mM borate buffer, pH 9, with a fixed concentration of enzyme i.e. 10,000
units/ml of Lipoxidase in 200 mM borate buffer, pH 9.0 in the presence and absence of most
8

active compound 33 (concluded from IC50 determination). The inhibitory kinetics were evaluated
by Lineweaver and Burk method [23]
3. Results and discussion
3.1. Chemistry
The synthetic protocols for the synthesis of target molecules of series 1 involve three steps.
Initially 7-hydroxy-4-methyl-2H-chromen-2-one (3) was likely formed via Pechmann reaction
between resorcinol (1) and ethyl acetoacetate (2) (Scheme 1). This step involves the interaction
of a phenol with a β-ketoester in the presence of an acid condensing agent. Concentrated
sulphuric acid was used as the condensing agent for simple monohydric phenols and β -
ketoesters. The mechanism of the reaction is thought to involve initial formation of a β-hydroxy
ester, which then cyclizes and dehydrates to yield the coumarin. In the second step, 4-methyl-7-
(oxiran-2-ylmethoxy)-2H-chromen-2-one (5) was synthesized by reacting 7-hydroxy-4-methyl-
2H-chromen-2-one (3) with excess of epichlorohydrin (4) in presence of K2CO3 under reflux
conditions. 7-(3-Chloro-2-hydroxypropoxy)-4-methyl-2H-chromen-2-one (5a) was obtained as a
side product in low yield in the synthesis of compounds. The epoxidation reaction was achieved
most conveniently by employing coumarin in a solvent such as chloroform, THF, under basic
condition. Various bases like NaOH and KOH were used but this resulted in ring opening, only
anhydrous K2CO3 afforded the desired intermediate product (5). Side product 5a in low yield was
separated using column chromatography over silica gel (60–120 mesh), and elution with 25%
ethyl acetate in hexane, 18% ethyl acetate in hexane furnished intermediate 5. The synthesis of
target compounds (7-28) (Scheme 1) (amino alcohol derivatives) of series 1 were accomplished
from the nucleophilic opening of oxirane with different anilines (6a-v) using ethanol under
refluxing condition. In series 2, a total number of 5 compounds were synthesized in two steps
(Scheme 2). In first step substituted benzothiazole derivatives (30a-e) were synthesized by
reacting substitiuted aniline (29a-e), and potassium thiocyanate in the presence of bromine in
glacial acetic acid. In second step, substituted benzothiazole derivatives (30a-e) were reacted
with 4-methyl-7-(oxiran-2-ylmethoxy)-2H-chromen-2-one (5) to afforded final compounds (31-
35) of series 2. The structure elucidation of synthesized compounds was carried out by FTIR,
1
13
mass spectroscopy, H and C NMR spectroscopy and elemental analysis. The FTIR spectra of
intermediate compounds showed the characteristic ketone peak in range of 1705-1745 cm-1
(Figure 1S and 4S under supplementary data). In FTIR spectrum of 7, peak of aromatic O-H
9

stretch was found around 3421 cm-1 and 3247 cm-1. A peak of CN was observed at 2250 cm-1
represents formation of coumarin ring. In mass spectra a peak at m/z 325.9 showed the molecular
ion peak, which represent [M+H]+ peak. The most commonly observed metal adduct ions of
charge sodium and potassium adducts in positive-ion electrospray are analyses at [M+Na]+ and
[M+K]+ (Figure 2S and 5S under supplementary data). As reported for the coumarin derivative
the base peak was observed at 150 m/z value with 100% intensity. This information indicated the
1
formation of the products with desired molecular mass. H NMR spectra showed a singlet peak
of methyl of coumarin occur at 1.7 δ value having integration value of 3 (Figure 3S and 6S
under supplementary data). A hydroxyl proton peak was found at 2 δ as doublet and as down
field due to its attachment to nearby oxygen and nitrogen doublet at 3.4 with integration value
around 2 which characterized the presence of -CH2. Another peak with integration value of 2 as
triplet proves another -CH2 proton. A triplet peak of amine at 4.1 δ was characterized by its
integration value 0.92. Also there are multiplets of the aromatic protons with their integration
value near to 1, and thus, it was accounted from the spectra that there are 9 aromatic protons in 7.
Some impurity peaks were found at region of 7.32 δ and 1.2 δ which might be due to CDCl3 and
CHCl3, respectively.
3.2 Molecular Modeling Study
3.2.1. Comparison of binding site of human 5-LOX with soybean 3-LOX
Binding site residues of human 5-LOX (PDB ID: 3v99), and soybean 3-LOX (PDB ID: 1ik3)
were identified through COFACTOR server. Results of COFACTOR server were submitted to
MultiBind server for pair wise alignments of human 5-LOX and soybean 3-LOX enzymes. Pair
wise surface alignment of both the enzymes detected eleven common physico-chemical features
(Fig. 1) (Table S1 under supplementary data). They are aligned with scores 29.555. The method
is based on a representation of each amino acid of an enzyme as a set of features that are
important for its interaction with other molecules. The binding site of 5-LOX and 3-LOX have
all eleven common features, which are represented by four PII (aromatic property through pi
contacts), three ALI (aliphatic hydrophobic property), two ACC (hydrogen bond acceptor), one
DON (hydrogen bond donor), and one DAC (hydrogen bond donor and acceptor) features (Table
S1 under supplementary data). Results of this comparison (Fig. 1), suggested that amino acid in
the binding sites of human 5-LOX share almost the same similarity with soybean 3-LOX
enzyme.
10

3.2.2. Docking results
In order to known the binding mode and interaction with amino acid residues of 5-lipoxygenase
(5-LOX) enzyme, we performed molecular docking study of synthesized compounds in the
binding site of 5-LOX. Gilbert et al. [24] solved the crystal structure of human 5-lipoxygenase
enzyme in the year 2011. Docking study further helped in the identification of active compounds.
Diclofenac sodium was used as the reference drug in pharmacological screening, so diclofenac
was also docked in 5-LOX enzyme as reference molecules. Docking results are shown in Table
1. Docking results showed that most of the compounds formed interactions with the key amino
acid residues like HIS372, GLY557, LYS409, GLN413, HIS550, ASN554, TYR558, and
ALA672 involved in the binding of inhibitors in the vicinity of active site. Series 2 compounds
showed highest gold docking score as compared to series 1 compounds. Compound 28 showed a
highest gold docking score of 74.143, and formed two H-bond with 5-LOX enzyme (Fig. 2A).
Oxygen atom in chromen ring act as H-bond acceptor and formed H-bond with GLY557 (2.8 Å)
and -OH group of propoxy linker formed H-bond with HIS372 (3.04 Å). Compound 33 showed
second highest docking score of 71.767, and formed three hydrogen bonds (Fig. 2B), ALA672
formed H-bond (2.71 Å) with -NH of 2-hydroxy aminopropoxy linker, oxygen atom of chromen
ring formed H-bond with GLY557 (2.75 Å) in similar way as compound 28, and third H-bond
was formed in between –OH group of 2-hydroxy aminopropoxy linker in 33, and HIS550 (3.04
Å). Compound 31 showed docking score of 71. 245 and formed five H-bond in the active site of
5-LOX (Fig. 2C). Reference compound diclofenac also formed two H-bond with the docking
score of 67.819 (Fig. 2D).
3.3. Pharmacological screening
All the synthesized compounds were screened for their in vivo anti-inflammatory activity by
carrageenan induced rat paw edema model, analgesic activity by acetic acid induced writhing
method, and radiant heat method (tail-flick method), and for ulcerogenic potential. The most
active compounds were evaluated in vitro for LOX inhibition. Diclofenac sodium was used as the
reference drug.
11

3.3.1. Anti-inflammatory activity
Mean changes in paw edema thickness of animals pretreated with the test compounds at 50
mg/kg body weight were measured after 1, 2, 3, and 6 h after induction of inflammation. One
way ANOVA followed by Tukey’s test was used to evaluated statistical significance and to
differences between control and treatment groups. Compounds 7, 8, 12, 14, 17 of series 1 and all
the compounds of series 2 (31-35) were found to possess anti-inflammatory activity, and showed
potent activity (68 to 80% protection form inflammation after 3 h). The results are shown in
Table 2 (Figure 7S and Table S3 under supplementary data). Replacement of substituted phenyl
ring attached with amino alcohol linker in series 1 with substituted benzothiazole ring with the
presence of bulky group (-OCH3) led to have potent inhibitory activity of the series 2
compounds. Among the active compounds of series 1, compounds with electronegative halogen
groups like 4–Cl (12), 2,6-diCl (14), and 4-F (17) showed more than 68% protection from
inflammation. Substitution pattern of mono and di-substituted electronegative –Cl group on
phenyl ring played an important role in determination of anti-inflammatory activity of
synthesized compounds. 2,6-diCl (14) and 4-Cl (12) substituted compounds were found activity,
whereas 3-Cl (11), 2,3-diCl (13), 3,5-diCl (15), and 3,4-diCl (16) were found inactive.
Compounds with 4-OCH3 (24), 3-OC2H5 (25), 4-C2H5Ph (27) groups on phenyl ring and,
compounds with electron donating groups like 3-NH2 (22) and 2-SH (23) were also found
inactive. Unsbstituted phenyl ring containing compound 7 and compound with 2-Me group on –
Ph ring (8) showed good anti-inflammatory activity.
3.3.2. Analgesic activity by acetic acid writhing method
All the synthesized compounds were further evaluated in vivo for antinociceptive potential using
acetic acid induced writhing method. Acetic acid induces the release of mediators of pain such as
PGs at the peritoneal receptors, which stimulates nociceptive neurons. Analgesic activity of
synthesized compounds on the acetic acid writhing method is summarized in Table 3 (Figure 8S
and Table S4 under supplementary data). All the compounds of series 2 (31-35) showed
analgesic activity. Compound 35 showed significant reduction in writhing responses, and
produced more than 60% protection from pain. Compound 33 also showed more than 55%
protection form pain. Compound 7, 14, 31, and 34 showed more than 34% protection form pain,
which is comparable with standard diclofenac sodium which showed 37.65% protection.
12

3.3.3. Analgesic activity by radiant heat method (Tail-flick method)
Synthesized compounds of series 2 (31-35) showed significant anti-inflammatory activity, and
antinociceptive potential were further selected for in vivo analgesic activity by Tail-flick method.
Out of the five synthesized compounds of series 2, 33 and 35 showed significant activity at 120
min (Fig. 3). Presence of –OCH3 group at -C6 position of benzothiazole ring in compound 35,
and electronegative –Cl in 33 were found very active substitution in determination of analgesic
activity. Di-substituted 32 (5-Cl,6-F) and 34 (5,6-diCl) compounds showed decreased activity as
compare to mono-substituted (-C6) 33 and 35. Results of analgesic activity revealed that the
presence of substitution at –C6 position of benzothiazole ring is crucial in determination of
analgesic activity.
3.3.4. Ulcerogenic risk evaluation
Synthesized compounds of series 2 (31-35) showed significant anti-inflammatory and analgesic
activity, and further tested for their ulcerogenic risk in term of presence and absence of gastric
irritation, and measurement of ulcerogenic area at a higher dose of 150 mg/kg, p.o. which is
three times of the dose used for anti-inflammatory and analgesic activity. Compounds 33 and 35
were found to be safer as gastric lesion risks at higher dose when compared to aspirin (Fig. 4).
The study revealed that these compounds causing less gastric lesions at higher dose as compared
to aspirin, may show reduced ulcerogenic potential at lower doses.
3.3.5. In-vitro LOX inhibition assay
The synthesized compounds with potent anti-inflammatory and analgesic activity were tested in
vitro for their LOX enzyme inhibition activity. Among the five tested compounds, 33 (IC50 =
2.79 nM), and 35 (IC50 = 2.09 nM) (Table 4) (Figure 9S under supplementary data) showed
potent inhibition of LOX enzyme. The LOX enzyme inhibition activity data revealed a clear
preference for the potency when–Cl group, and –OCH3 group at -C6 position of benzothiazole
ring were substituted. Di-substituted 32 (5-Cl,6-F) and 34 (5,6-diCl) showed less LOX enzyme
inhibition activity as compare to mono-substituted (-C6) 33 and 35. LOX enzyme inhibition
activity data is in agreement with docking results, and anti-inflammatory and analgesic activity,
and showed similar structure activity relationship pattern as found in anti-inflammatory and
analgesic activity.
13

3.3.6 In-vitro kinetic study
In order to establish the mode of enzyme inhibition, compound 35 was selected for LOX-
inhibition kinetic study using Lineweaver-Burk reciprocal plots. The plot was graphed as the
reciprocal of substrate concentration versus reciprocal of reaction rate (in presence and absence
of inhibitor) (Fig. 4). The results obtained from the kinetic study showed increased slope in
presence of inhibitor (35), which ultimately indicated decrement in the Vmax level without any
significant alteration in Km value. The kinetic inhibition pattern of 35 showed mixed or non-
competitive type of inhibition with 5-LOX enzyme.
4. Conclusion
In this study, 7-subsituted coumarin derivatives were synthesized, and evaluated in vivo for anti-
inflammatory and analgesic activity, and ulcerogenic risk potential. The most active compounds
were evaluated in vitro for lipoxygenase (LOX) inhibition. A total number of 27 compounds
1
were synthesized in good yield. The synthesized compound were characterized by FTIR, H and
13C NMR, mass spectra (ESI-MS), and elemental analysis. In vivo anti-inflammatory and
analgesic activity, and in vitro LOX enzyme inhibition study revealed that compound 33 and 35
were found as the most potent compounds in all the screening methods. In vitro kinetic study of
35 showed mixed or non-competitive type of inhibition with LOX enzyme. When these results
are taken together, presence of -OCH3 group in 35 and electronegative -Cl group in 33 at C6-
position of benzothiazole ring were found more potent. Presence of benzothiazole ring in series 2
compounds as compared to substituted phenyl ring in series 1 compound with amino alcohol
linker at 7th-position of coumarin ring played critical role in interaction with 5-LOX enzyme in
docking study as well as in all the pharmacological screening methods.
Acknowledgment
The authors thank to Nirma University, Ahmedabad, Gujarat India for providing necessary
facilities to complete this work.
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16

Scheme and Figure captions
Caption to Scheme 1
Scheme 1 Synthesis of compounds 7-28. Reagents and conditions: (a) H2SO4; (b) K2CO3; (c)
C2H5OH
Caption to Scheme 2
Scheme 2 Synthesis of compounds 31-35. Reagents and conditions: (d) Br2 in CH3COOH; (e)
C2H5OH
Caption to Fig. 1
Fig. 1 Comparison of binding site of human 5-LOX with soybean 3-LOX. (A) Pair wise surface
alignment of amino acids of binding site of human 5-LOX (blue coloured) and soybean 3-LOX
(red coloured). (B) Common physiochemical features identified for human 5-LOX, and soybean
3-LOX.
Caption to Fig. 2
Fig. 2 Binding mode of compound 28 (A), 33 (B), 31 (C), and diclofenac (D) in 5-LOX enzyme
proposed by docking studies. Compounds are in ball and stick model with color by atoms, H-
bond interaction was highlighted using green dotted lines. The labeled protein residues are in
capped stick model with color by atom.
Caption to Fig. 3
Fig. 3 Anti-nociceptive potential of synthesized compounds. Reaction time after treatment with
test compounds 60 min (A), 90 min (B), 120 min (C). Values are mean ± SEM, (n = 6)
Caption to Fig. 4
Fig. 4 Ulcerogenic area documented in stomach linings, Values are mean ± SEM, (n = 4)
Caption to Fig. 5
Fig. 5 Plot showing mixed inhibition of 5-LOX enzyme by compound 35
17

Table 1
Docking score with binding site interacting amino acid residues
Sr. no. Compound Amino acid residue involved in H-bond formation
Docking
score
1
HIS372, GLY557
74.143
71.767
71.245
70.658
70.111
69.102
68.618
67.260
66.784
65.679
65.653
65.522
65.422
64.658
64.530
64.323
63.946
63.811
63.623
62.672
62.309
62.096
60.016
59.027
58.680
58.282
57.986
67.819
28
33
31
35
27
20
32
15
26
11
20
23
9
2
HIS372, GLY557, ALA672
HIS372, GLY557, ALA672, HIS550, LYS409
GLN413, TYR558
HIS372, GLY557
3
4
5
6
HIS372, GLY557, ALA672
ASN554 , GLN413
GLN557
7
8
9
HIS372, GLY557, ALA672, LYS409
HIS372, GLY557, ALA672
HIS372, GLY557, ALA672
GLY557, ALA672
GLN557, HIS550
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
GLN557, HIS550, HIS372
GLN557, HIS372
13
18
19
22
12
25
17
24
16
10
8
GLN557, HIS372, ALA672
GLN557, ALA672
GLN557
GLN557, HIS372
GLN557, HIS372
ASN554
GLY557, ALA672
ASN554
ASN554
HIS372, GLY557, ALA672
GLN557, HIS372
14
21
7
GLN557
Diclofenac HIS550, VAL671
16

Table 2
Anti-inflammatory activity of synthesized compounds, diclofenac sodium and control (50
mg/kg p.o.) against carrageenan induced paw edema in rats
Compound
Mean Protection (%)
1 h
2 h
3 h
6 h
32.66
70.54
65.56
69.54
69.99
65.20
75.42
64.12
70.35
64.48
75.15
72.41
63.34
7
36.04
49.32
33.34
32.43
46.85
32.88
30.18
32.66
54.05
68.19
72.63
68.34
72.56
76.34
67.20
67.98
66.77
80.99
57.31
67.14
54.50
59.68
63.49
59.75
49.50
63.19
64.56
8
12
14
17
31
32
33
34
35
Control
0
0
0
0
Diclofenac
68.47
85.90
89.28
77.18
sodium (Std)
One way ANOVA followed by Tukey’s test, p < 0.05 vs control group
Values are mean ± SEM, (n = 6)
17

Table 3
Analgesic activity of synthesized compounds using acetic acid writhing method
Compound
% Protection
37.04
7
28.09
25.93
37.35
28.70
34.26
25.00
55.25
38.58
60.80
8
12
14
17
31
32
33
34
35
Control
0.00
Diclofenac sodium (Std)
37.65
One way ANOVA followed by Tukey’s test, p < 0.05 vs control group
18

Table 4
IC50 values describing the effect of synthesized compounds (31- 35) on 5-LOX enzyme
Compound
IC50 (nM)
3.239 ± 0.575
31
32
33
34
35
6.157 ± 0.541
2.792 ± 0.243
15.503 ± 2.553
2.118 ± 0.024
Results are means of three determinations
19

GRAPHICAL ABSTRACT
Synthesis, anti-inflammatory, analgesic, 5-lipoxygenase (5-LOX)
inhibition activities and molecular docking study of 7-substituted
coumarin derivatives
Pavan Srivastavaa, Vivek K. Vyasa, BhaveshVariyab, Palak Patelc, Gulamnizami Qureshia,
Manjunath Ghatea*
aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University,
Ahmedabad 382481, Gujarat, India
bDepartment of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad 382481,
Gujarat, India
cInstitute of Science, Nirma University, Ahmedabad 382481, Gujarat, India
18