Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2016.02.025

Secretory phospholipase A₂ (sPLA₂) is an important enzyme that plays a key role in various inflammatory diseases including cancer and its inhibitors have been developed as preventive or therapeutic agents. In the present study, a series of new indole containing isoxazole derivatives (10a–10o) is synthesized and evaluated for their sPLA₂ inhibitory activities. All compounds (10a–10o) showed significant sPLA₂ inhibition activities both in vitro and in vivo studies which is substantiated in in silico studies. Among all the tested compounds, 10o showed potent sPLA₂ inhibition activity, that is comparable or more to ursolic acid (positive control). Further studies demonstrated that 10o showed in vitro antiproliferative activity when tested against MCF-7 breast and DU145 prostate cancer cells. Furthermore, compounds 10a–10o obeyed lipinsky's rule of 5 and suggesting druggable properties. The in vitro, in vivo and in silico results are encouraging and warrant pre-clinical studies to develop sPLA₂-inhibitory compound 10o as novel therapeutic agent for various inflammatory disorders and several malignancies.

Full text of DOI:10.1016/j.ejmech.2016.02.025

Accepted Manuscript
Synthesis of new secretory phospholipase A -inhibitory indole containing isoxazole
2
derivatives as anti-inflammatory and anticancer Agents
Srinivasa Rao Pedada, Nagendra Sastry Yarla, Pawan J. Tambade, Dhananjaya
Bhadrapura Lakkappa, Anuapam Bishayee, Kalle M. Arunasree, Gundala Harold
Philip, Gangappa Dharmapuri, Gjumrach Aliev, Swathi Putta, Gururaja Rangaiah
PII:
S0223-5234(16)30095-2
10.1016/j.ejmech.2016.02.025
EJMECH 8376
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 September 2015
Revised Date: 7 February 2016
Accepted Date: 8 February 2016
Please cite this article as: S.R. Pedada, N.S. Yarla, P.J. Tambade, D.B. Lakkappa, A. Bishayee, K.M.
Arunasree, G.H. Philip, G. Dharmapuri, G. Aliev, S. Putta, G. Rangaiah, Synthesis of new secretory
phospholipase A -inhibitory indole containing isoxazole derivatives as anti-inflammatory and anticancer
2
Agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.025.
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ACCEPTED MANUSCRIPT
Graphical abstract
O
N
O
N
R
R
HN
HN
R1
R1
N
Boc
R2
N
R2
H
10o (R1=F, R2=CF3, R=CH3)
GIIA sPLA2 (IC50 = 10.23±0.91 µM)

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Synthesis of new secretory phospholipase A2-inhibitory indole containing isoxazole derivatives
as anti-inflammatory and anticancer Agents
Running title: sPLA2-inhibitory indole containing isoxazoles
Srinivasa Rao Pedadaa, +, δ, Nagendra Sastry Yarlab, δ, Pawan J. Tambadec, Dhananjaya Bhadrapura
Lakkappad, Anuapam Bishayeee, Kalle M. Arunasreef, Gundala Harold Philipg, Gangappa
Dharmapurif, g, Gjumrach Alievh, Swathi Puttai, Gururaja Rangaiah j,*
aCentre for Research and development,PRIST University, Thanjavur-613 403, Tamilnadu, India
bDepartment of Biochemistry, Institute of Science, GITAM University, Visakhapatnam-530 045,
Andhra Pradesh, India
cDepartment of Chemistry, Arts, Commerce and Science College, Nandgaon, Maharashtra, India
dToxinology/Toxicology and Drug Discovery Unit, Center for Emerging Technologies, Jain Global
Campus, Jain University, Ramanagara-562 112, Karnataka, India
eDepartment of Pharmaceutical Sciences, College of Pharmacy, Larkin Health Sciences Institute,
Miami, Florida 33169, USA
fDepartment of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad-500
046, Telangana, India
gDepartment of Biotechnology, Sri Krishnadevaraya University, Anantapuramu-515 003, Andhra
Pradesh, India
hSchool of Health Sciences and Healthcare Administration, University of Atlanta, E. Johns
Crossing, Johns Creek, GA 30097, USA
iPharmacology Division, University College of Pharmaceutical Sciences, Andhra University,
Visakhapatnam-530003, Andhra Pradesh, India
jDepartment of Biochemistry, Sarada Vilas College, Mysore-570004, Karnataka, India
+the work is part of PhD work
δ equally contributed to this work
*corresponding author. E-mail address: dr.gururaja@gmail.com (G. Rangaiah)
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ABSTRACT
Secretory phospholipase A2 (sPLA2) is an important enzyme that plays a key role in various
inflammatory diseases including cancer and its inhibitors have been developed as preventive or
therapeutic agents. In the present study, a series of new indole containing isoxazole derivatives
(10a-10o) is synthesized and evaluated for their sPLA2 inhibitory activities. All compounds (10a-
10o) showed significant sPLA2 inhibition activities both in vitro and in vivo studies which is
substantiated in in silico studies. Among all the tested compounds, 10o showed potent sPLA2
inhibition activity, that is comparable or more to ursolic acid (positive control). Further studies
demonstrated that 10o showed in vitro antiproliferative activity when tested against MCF-7 breast
and DU145 prostate cancer cells. Furthermore, compounds 10a-10o obeyed lipinsky`s rule of 5 and
suggesting druggable properties. The in vitro, in vivo and in silico results are encouraging and
warrant pre-clinical studies to develop sPLA2-inhibitory compound 10o as novel therapeutic agent
for various inflammatory disorders and several malignancies.
Key words:
Indole, Isooxazole, Secretory phospholipase A2, Anticancer, Anti-inflammatory activity
2

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1.
Introduction
Phospholipase A2s (PLA2s) are group of enzymes, which cleave phospholipids specifically
at sn-2 position to liberate free fatty acid, mostly arachidonic acid (AA) and lysophospholipids
(LPLs) [1-3]. Inhibition of PLA2 prevents the liberation of AA and LPLs. Hence, researchers have
been considering that PLA2s could be good therapeutic target than cyclooxygense-2 and
lipoxygenase, which are downstream enzymes [1-3]. Several isoforms of PLA2s exist, which are
divided into various groups; secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and calcium-
independent PLA2 (iPLA2), platelet activating factor-acyl hydrolase (PAF-AH), lysosomal PLA2
(LPLA2), adipose-specific PLA2 (AdPLA2). The secreted PLA2s (13-55 kDa) are the first type of
PLA2 enzymes discovered and categorized into several groups [4]. Among all groups of the sPLA2s,
group IIA sPLA2 (GIIA sPLA2) is predominantly distributed and plays key role in various
inflammatory diseases, including arthritis, inflammatory bowel disease, atherosclerosis, psoriasis,
and several cancers [1-4].
Realizing the role of sPLA2 in inflammatory diseases and various cancers, considerable
efforts are being made by several investigators for the discovery and development of sPLA2
inhibitors as therapeutic or preventive agents [2]. Several sPLA2 inhibitors have been developed and
some of the sPLA2 inhibitors are currently under clinical trials for various inflammatory and
oncologic diseases [2-4]. During last several decades, various derivatives of indoles have been
developed as GIIA sPLA2-inhibitors [5-7]. A series of indole acids, indole amides, indole-3-
acetamides, indole-3-glyoxamides are developed as sPLA2 inhibitors [5]. Lilly Research Laboratory
(Indianapolis, IN, USA) developed a series of indole-3-acetamide derivatives with sPLA2 inhibition
activity. Among all compounds, varespladib or LY315920 inhibited group IIA sPLA2 with high
potency and it entered into clinical trials for inflammatory diseases [6]. However, LY315920 or
varespladib failed phase II clinical trials because of poor anti-inflammatory efficacy. Further, Lilly
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Research Laboratory synthesized LY333013 or methyl varespladib, a modified form to LY315920,
but it also failed in clinical trials due to its poor anti-inflammatory efficacy [7]. Recently, Anthera
Pharmaceuticals (Hayward‚ CA, USA) announced the use of A-001 (varespladib or LY315920) and
A-002 (LY333013 or methyl varespladib) in treatment of cardiovascular diseases as their efficacy in
reducing LDL-C and atherosclerosis in patients with acute coronary syndrome (ACS) along with
atorvastastin treatment, according to phase II clinical trial namely, Fewer Recurrent Acute Coronary
Events With Near Term Cardiovascular Inflammation Suppression (FRANCIS) [8]. In phase III
clinical trial of varespladib in patients with ACS, namely Vascular Inflammation Supression to
Treat Acute Coronary Syndrome for 16 days (VISTA-16), varespladib showed poor efficacy in
reduction of ACS and significantly increased the risk of nonfatal myocardial infarction (MI) [9].
Most of the sPLA2 inhibitors failed in clinical trials due to poor efficacy and adverse effects [2].
Therefore, discovery of new sPLA2-inhibitory molecules or optimization of existed sPLA2-
inhibitory compounds with more potency, safety and selectivity is urgently needed for prevention
and treatment of inflammatory disorders and several cancers.
Isoxazoles have been reported to possess various biological activities, including PLA2-
inhibitory activities [10, 11]. Sadashiva et al. [11] synthesized trimethoxyphenyl isoxazolidine
derivatives with sPLA2 inhibitory activities. Various derivatives of indoles have been developed as
sPLA2 inhibitors and some of them are under clinical evaluation for various inflammatory diseases.
Both indoles and isoxazoles are attractive chemical scaffolds for development of novel sPLA2
inhibitors [5-7, 10, 11]. Considering this background, the present study aimed to synthesize indole
containing isoxazoles (10a-10o) with potent sPLA2-inhibitory activity. Among all compounds, 10o
showed substantial sPLA2-inhibitory activity. Further in vivo antiinflammatry studies of 10o were
performed using inflammogen (carrageenan)-induced rat paw edema model [12]. In vitro anticancer
studies using sPLA2-inhibitory 10o were also performed on MCF-7 breast carcinoma and DU145
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prostate cancer cell lines since sPLA2 exhibited pro-tumorigenic role in breast and prostate cancers
[13, 14].
2.
2.1. Chemistry
The target molecules were synthesized in a convergent fashion by coupling indole aldehydes
Results
8(a-c) and isoxazole containing primary amines 5(a-e), which were subsequently converted to their
hydrochloride salts 10 (a-o). The isoxazole methyl amines were synthesized starting from
commercially available aromatic aldehydes. Aldehydes were reacted with hydroxyl amine
hydrochloride to obtain their corresponding oxime derivatives 2(a-e). They were converted to their
corresponding bromo methyl isoxazoles by intermolecular cycloaddition of activated oximes with
with propargyl bromide in presence of chloramine-T in ethanol [15]. The bromo methyl isoxazoles
were later converted to azides by treating with sodium azide in DMF 4(a-e), which were finally
reduced to the desired isoxazole methyl amines 5(a-e) by following a mild and efficient
Schodinger’s protocol using triphenyl phosphine (TPP) and water in THF as a solvent system [16].
On the other hand indole aldehydes were synthesized by electrophilic acylation of
unprotected indoles following Vilsmier-Haack method using POCl3/DMF in good to excellent
yields 7(a-c). To avoid any unforeseen complications, the indole NH was protected by Boc
anhydride. The boc protected indole-3-aldehydes 8(a-c) and isoxazole methyl amines 5(a-e) were
coupled by reductive amination in the presence of sodium cyanoborohydride and catalytic amount
of acetic acid in methanol at ambient temperatures. The isolated yields of the coupled products 9(a-
o) were moderate to good. Finally, Boc group was deprotected with TFA in DCM, which were
subsequently converted to their hydrochloride salts with 20% HCl in ethyl acetate in good yields
10(a-o) (Scheme-1). All the synthesized compounds were well characterized by 1H NMR, 13C NMR,
IR and mass spectra.
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2.2.
Pharmacology
2.2.1. sPLA2-inhibitory activity of indole containing isoxazole derivatives (10a-10o)
All synthesized indole containing isoxazole derivatives showed significant in vitro sPLA2-
inhibitory activity and good binding affinity on crystal structure of sPLA2 (Table 1). Among all
tested compounds, 10o showed substantial sPLA2-inhibitory activity with IC50 of 10.23±0.91 ꢀM in
vitro and docked on crystal structure of sPLA2 with binding energy of -129.5 Kcal/mol where as
ursolic acid (positive control) inhibited with IC50 of 12.59±1.03 ꢀM in vitro and docked with
binding energy of -128.8 Kcal/mol (Fig. 1 & Table 1). Both in vitro and in silico results
demonstrated that 10o showed comparable or more sPLA2-inhibitory activity than ursolic acid
(positive control). Further in silico studies demonstrated that 10o binds the vicinity of amino acid
residues at active site PHE-5, TYR-21, GLY-22, CYS-28, GLY-29, CYS-44, HIS-47, ASP-48,
TYR-51, and PHE-98 (Fig. 2). The strong binding affinity and interactions of 10o with sPLA2 may
be responsible for its sPLA2-inhibitory activity. From the Table 1 it was evident that compounds
containing electron donating group such as CH3 on indole ring (R group) and electron withdrawing
groups, such as F, CF3, and NO2 on isoxazole phenyl ring (R1, R2 group) are found to have
remarkable activities, which were further supported by structure activity relationship (SAR) studies.
When R1 and R2 = H, R= CH3>H>Br.
2.2.2. Antiiflammatory activity of 10o in wistar albino rats
In vivo oral anti-inflammatory efficacy of 10o (10 mg/kg body weight) was tested on group
of rats using carrageenan model and paw volumes (ml) of treated 10o and ursolic acid (positive
control)) and untreated (vehicle control) (Fig. 3). 10o (10 mg/kg body weight) showed 75.67±4.21%,
and 76.54±3.1% edema inhibition (maximum) at 3rd h and 4th h, respectively, where as ursolic acid
(10 mg/kg body weight) showed 72.97±3.56% and 75.17±5.70% edema inhibition at 3rd h and 4th h,
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respectively. 10o exhibited in vivo anti-inflammatory activity wich was comparable to or more than
ursolic acid (positive control).
2.2.3. Antiproliferative activity of 10o against breast and prostate cancer cells
Antiproliferative effect of 10o was evaluated against MCF-7 breast and DU145 prostate
cancer cell lines by MTT assay. Compound 10o showed significant anti-proliferative effect in
MCF-7 and DU145 cancer cell lines with IC50 of 10.03±0.86 and 14.15±1.2 µM, respectively,
whereas doxorubicin (positive control) exhibited anticancer activity with 8.02±0.69 and 4.75±0.35
µM on respective cancer cell lines (Fig. 4). The results demonstrate that 10o showed comparable
antiproliferative effect against MCF-7 breast cancer cells with that of doxorubicin (positive control).
2.2.4. Drug likeness of compounds
In addition to sPLA2 inhibition studies, the evaluation of druglikeness of compounds
according to Lipinski’s rule of five is done which states that, in general, an orally active drug has
not more than 5 hydrogen bond donors (OH and NH groups), not more than 10 hydrogen bond
acceptors (notably N and O), molecular weigh under 500 g/mol, partition coefficient log P less than
5. Almost all compounds 10a-10o obey lipinsky`s rule and is of druggable nature (Table 2).
3. Discussion
Several sPLA2 inhibitory molecules have been developed as antinflammaotry and anticaner
agents by several investigation and some of them under pre-clinical and clinilcal developmental
stage. Mayur et al. [17] synthesized azetidin-2-one derivatives wth sPLA2 inhibitory activity.
Further studies demonstrated that azetidin-2-one derivatives showed in vivo anti-inflammatory
activity in mice. 4-tetradecyloxybenzamidine (PMS815) derivatives were designed and synthesized
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with PLA2 inhibitory activity [18]. Among all compounds, 4,5-dihydro-3-(4-tetradecyloxybenzyl)-
1,2,4-4H-oxadiazol-5-one (PMS1062) exhibited a micromolar IC50 towards GII sPLA2s, while
inactive towards GI and GIII PLA2s in vitro and also blocked the GII sPLA2 activities induced by
lipopolysaccharides and interleukin-6 in HepG2 cell line and no cytotoxicity was observed in LLC-
PK1 and A549 cell lines [18]. Several indole derivatives have been developed as sPLA2 inhibitors
and some of them are entered into clinical trials for various inflammatory diseases [2, 3, 5-7].
Sadashiva et al. [11] synthesized trimethoxyphenyl isoxazolidine derivatives with sPLA2 inhibitory
activities. Indole and isoxazoles are attractive scaffolds for development of novel sPLA2 inhibitors.
Considering this background, in the present study, we have synthesized indole containing isoxazole
derivatives and evaluated for their sPLA2 inhibitory activity. Among all compounds, 10o showed
substantial sPLA2 inhibitory activity. In in vivo anti-inflammatory studies, 10o exhibited anti-
inflammatory activity wich was comparable to or more than ursolic acid (positive control). In
anticancer studies, 10o showed comparable antiproliferative effect against MCF-7 breast cancer
cells with that of doxorubicin (positive control). Moreover all compounds including 10o obey
Lipinsky`s rule of 5 and can be druggable. Further detailed investigations are needed to develop
sPLA2-inhibitory 10o as antiinflammatory and anticancer agent upon encouraging resutls of in vitro
and in vivo studies.
4.
Conclusions
In the present study, a series of novel indole containing isoxazole derivatives (10a-o) were
synthesized and showed significant sPLA2-inhibitory activities. Remarkable sPLA2 inhibition
activities were observed when electron donating group like -CH3 on indole ring and electron
withdrawing groups like -F, and -CF3 on phenyl ring of isoxazole were present. Among all
compounds, 10o showed substantial sPLA2-inhibitory activity. 10o also showed significant in vivo
anti-inflammatory activity in carrageenan-induced rat paw edema model as well as good
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antiproliferative effect against breast MCF-7 and prostate DU145 cancer cells. Moreover,
compounds 10a-10o obey Lipinsky`s rule of 5 and can be druggable. The in vitro, in silico and in
vivo results of this study are encouraging and warrant further investigations to develop sPLA2-
inhibitory compound 10o as novel therapeutic agent for inflammatory disorders and oncologic
diseases.
5.
Experimental
5.1.
Chemistry
5.1.1. General information
All chemicals used for the synthesis were of reagent grade and procured from Sigma-
1
13
Aldrich, St. Louis, MO, USA. H and C NMR spectra were recorded on AS 400 MHz Varian
NMR spectrometer using TMS as an internal standard. IR spectra were recorded by using Perkin-
Elmer Spectrum 100 Series FT-IR spectrometer. Mass spectra were recorded on Agilent 1200
Series LC/MSD VL system. Melting points were determined by using Buchi melting point B-545
instrument and are uncorrected. All the reactions were monitored by thin layer chromatography
(TLC) using percolated silica 60 F254, 0.25 mm aluminum plates (Merck). The crude compounds
were purified by column chromatography using silica gel (100-200 mesh) and gradient (0-20%)
ethyl acetate in hexane as the eluent system.
5.1.2. General Procedure for the synthesis of 9(a-o)
To the stirred mixture of substituted indole (8(a-c), 0.50 g, 2.0 mmol) and substituted
isoxazole amine (5(a-e), 0.35 g, 2.0 mmol) in methanol (5.0 ml) was added 4A° molecular sieves
(0.50 g) and catalytic amount of acetic acid. The above reaction mixture was allowed to stir for 1h
at room temperature. followed by the portion wise addition of sodium cyanoborohydride (0.32 g,
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5.0 mmol) was added portionwise at room temperature and the reaction mixture was allowed to stir
for 16 h. Solvent was removed under reduced pressure and then diluted with dichloromethane,
which was washed with 10% sodium bicarbonate solution. The obtained DCM layer was dried over
sodium sulphate and distilled under vacuum to obtain crude 9(a-o), which was further purified by
column chromatography using ethyl acetate and hexane mixture (80:20) to obtain pure compounds
in 80% yield.
5.1.3. General Procedure for the synthesis of 10(a-o)
To a solution of compound 9(a-o) (0.5 g, 1.2 mmoles) in ethyl acetate (5.0 ml) was added
HCl in ethyl acetate (20%) by drop wise manner and stirred for 16h. The so obtained solid was
collected by filtration and washed with ethyl acetate, dried under vacuum to obtain compound 10(a-
o) in 63 – 79 % yield.
5.1.3.1. N-((1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine hydrochloride(10a)
Yield: 79 %; light orange solid; m.p. 185 – 187 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 4.45
(d, 4H, J = 10 Hz), 7.09 (dd, 1H, , J = 1.2 Hz, J = 8 Hz), 7.16 (dd, 1H, , J = 1.2 Hz, J = 8.0 Hz),
7.21 (s, 1H), 7.44 (d, 1H, J = 8.0 Hz) 7.58 – 7.52 (m, 3H), 7.64 (d, 1H, J = 2.8 Hz), 7.78 (d, 1H, J =
8.0 Hz) 7.90 – 7.80 (m, 2H), 9.92 (s, 2H), 11.47 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.3,
41.8, 104.5, 104.7, 112.2, 119.1, 119.6, 122.1, 127.0 (2C), 127.2, 128.3, 128.5, 129.7 (2C), 131.0,
136.4, 162.5, 165.34; IR (KBr) cm-1 : 3645, 3301, 3116, 2948, 2730, 2603. 2422, 1709, 1612, 1579,
1458, 1409, 1367, 1344, 1279, 1235, 1180, 1097, 1018, 947, 920, 825, 768, 734, 690; MS (ESI) m/z
[M-H]-:338.4(HCl salt); Anal. Calcd. for C19H18ClN3O; C, 67.15; H, 5.34; Cl, 10.43; N, 12.37; O,
4.71 %. Found C, 67.45; H, 5.34; N, 12.33 %.
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5.1.3.2. N-((5-bromo-1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine
hydrochloride
(10b)
Yield: 70 % , off white solid; m.p. 191 - 193 ˚C ; 1H NMR (400 MHz, DMSO-d6) δ: 4.46 (d,
4H, J = 15.6 Hz), 7.25 – 7.24, (m, 1H) 7.27 (d, 1H, , J = 4 Hz), 7.41 (s, 1H, , J = 8 Hz), 7.60 – 7.50
(m, 3H) 7.66 (d, 1H, , J = 4 Hz), 7.86 (dd, 2H, , J = 4 Hz, J = 8 Hz), 8.04 (d, 1H, J = 1.6 Hz) 9.78(s,
2H), 11.65 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.6, 104.5, 104.6, 112.4, 114.2, 121.8,
124.5, 124.6, 127.0, 128.5, 129.0, 129.7 (2C), 130.0, 130.9, 135.1, 162.5, 165.3; IR (KBr) cm-1 :
2947, 2809, 2697, 1695, 1604, 1526, 1466, 1388, 1348, 1281, 1230, 1172, 1093, 1039, 1017, 988,
953, 905, 853, 823, 802,740; MS (ESI) m/z [M-H]-: 380.2 Anal. Calcd. for C19H17BrClN3O; C,
54.50; H, 4.09; Br, 19.08; Cl, 8.47; N, 10.04; O, 3.82 %. Found C, 54.32; H, 4.27; N, 10.76 %.
5.1.3.3. N-((5-methyl-1H-indol-3-yl)methyl)(3-phenylisoxazol-5-yl)methanamine
hydrochloride
(10c) :
Yield: 65 %; light pink solid, m.p. 208 - 210 ˚C ;1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s,
3H), 4.45 (d, 4H, J = 20.8 Hz), 6.96 (dd, 1H, , J = 4.0 Hz, J = 8.0 Hz), 7.26 (s, 1H), 7.32 (d, 1H, , J
13
= 8.0 Hz) 7.57 – 7.53 (m, 5H), 7.87 – 7.84, (m, 2H), 9.87 (s, 2H), 11.34 (s, 1H); C NMR (100
MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 104.0, 104.5, 111.9, 118.6, 123.6, 124.6, 127.0, 127.4, 128.1,
128.3, 128.5, 129.7 (2C), 130.9, 134.7, 162.5, 165.3; IR (KBr) cm-1 : 2948, 2809, 2697, 1695, 1604,
1526, 1466, 1388, 1348, 1281, 1230, 1172, 1094, 1017, 953, 905, 850, 824, 802,740; MS (ESI) m/z
[M-H]-: 351.9 (HCl salt); Anal. Calcd. for C20H20ClN3O; C, 67.89; H, 5.70; Cl, 10.02; N, 11.88; O,
4.52 %. Found C, 67.67; H, 5.83; N, 11.74 %.
5.1.3.4. N-((1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanamine hydrochloride (10d)
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Yield: 70 %; off white solid; m.p. 191 – 193 ˚C;1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s,
3H), 4.45 (d, 4H, J = 20.0 Hz), 7.09 (t, 1H, J = 8.0 Hz), 7.14 (t, 1H, J = 8.0 Hz), 7.22 (s, 1H), 7.34
(d, 2H, J = 8.0 Hz) 7.44 (d, 1H, J = 8.0 Hz) 7.63 (d, 1H, J = 2.0 Hz), 7.74 (d, 2H, J = 8.0 Hz) 7.78
(d, 1H, J = 8.0 Hz), 9.95(s, 2H), 11.48 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 21.4, 40.5, 41.8,
104.3, 104.6, 112.2, 119.1, 119.6, 122.0, 124.5, 125.7, 126.91, 127.2, 128.3, 130.2 (2C), 136.3,
140.7, 162.4, 165.1; IR (KBr) cm-1 : 3556, 3306, 3112, 2936, 2722, 2608, 2411, 1725, 1613, 1571,
1464, 1357, 1268, 1224, 1182, 1094, 1016, 986, 946, 816, 754; MS (ESI) m/z [M-H]-; 351.9 (HCl
salt); Anal. Calcd. for Anal. Calcd. for C20H20ClN3O; C, 67.89; H, 5.70; Cl, 10.02; N, 11.88; O,
4.52 %. Found C, 67.67; H, 5.91; N, 11.92 %.
5.1.3.5. N-((5-bromo-1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanaminehydrochloride
(10e)
Yield: 70 %; off white solid; m.p. 197 - 199 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.42 (s,
3H), 4.43 (d, 4H, J = 12.0 Hz), 7.21 (s, 1H), 7.25 (dd, 1H, J = 1.6 Hz, J = 8.4 Hz), 7.35 (d, 2H, J =
8.0Hz), 7.41 (d, 1H, J = 8.0 Hz), 7.65 ( d, 1H, J = 4Hz),7.75 (d, 2H, J = 8.0 Hz), 8.04 (d, 1H, J = 4
Hz) 9.81 (s, 2H), 11.66 (s, 1H); 13C NMR (100 MHz, DMSO) δ: 21.4, 40.6, 41.6, 104.3, 104.6,
112.4, 114.2, 121.8, 124.5, 124.6, 125.7, 126.9, 129.0, 129.9, 130.2 (2C), 135.1, 140.7, 162.4,
165.1; IR (KBr) cm-1 : 2917, 2756, 2601, 2420, 1742, 1612, 1565, 1455, 1420, 1336, 1245, 1217,
1170, 1100, 1014, 914, 862, 845, 799, 723; MS (ESI) m/z [M-H]- 429.8 (HCl salt) ; Anal. Calcd. for
C20H19BrClN3O; C, 55.51; H, 4.43; Br, 18.46; Cl, 8.19; N, 9.71; O, 3.70%. Found C, 55.21; H,
4.73; N, 9.49 %.
5.1.3.6.
N-((5-methyl-1H-indol-3-yl)methyl)(3-p-tolylisoxazol-5-yl)methanaminehydrochloride
(10f)
12

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Yield: 63 %; off white solid; m.p. 185 - 187 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.49 (s,
6H), 4.43 (d, 4H, J = 16 Hz), 6.97 (dd, 1H, J = 1.6 Hz, J= 8.0 Hz), 7.21 (s, 1H), 7.37 – 7.32 (m, 3H),
13
7.56 – 7.53 (m, 2H), 7.75 (d, 2H, J = 8.0 Hz), 9.79 (s. 2H), 11.32 (s, 1H); C NMR (100 MHz,
DMSO-d6) δ: 21.4, 21.7, 40.5, 41.8, 104.0, 104.4, 111.9, 118.6, 123.6, 124.6, 125.7, 126.9, 127.4,
128.1, 128.3, 130.2 (2C), 134.7, 140.7, 162.4, 165.1; IR (KBr) cm-1 : 3118, 2955, 2718, 2609, 2440,
1709, 1612, 1593, 1463, 1368, 1344, 1282, 1233, 1174, 1091, 1020, 953, 925, 808, 769, 742, 691;
MS (ESI) m/z [M-H]- 367.1 (HCl salt) ;Anal. Calcd. for C21H22ClN3O; C, 68.56; H, 6.03; Cl, 9.64;
N, 11.42; O, 4.35%. Found C, 68.46; H, 6.23; N; 11.53 %.
5.1.3.7. N-((1H-indol-3-yl)methyl)(3-(4-methoxyphenyl)isoxazol-5-yl)methanaminehydrochloride
(10g)
Yield: 70 % ; off white solid; mp 184 – 186 ˚C; 1H NMR (400 MHz, DMSO-d6) δ: 3.82 (s,
3H), 4.43 (s, 4H), 7.10 (m, 3H), 7.18 (m, 2H), 7.44 (d, 1H, J = 8.0 Hz), 7.63 (d, 1H, J = 2.4 Hz),
7.78 (m, 3H), 9.91 (s, 2H), 11.47 (s, 1H,); 13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.8, 55.7,
104.2, 104.6, 112.2, 115.1 (2C), 119.1, 119.6, 120.8, 122.0, 127.2, 128.3, 128.5 (2C), 136.4, 161.3,
162.1, 164.9; IR (KBr) cm-1 : 3440, 3131, 2936, 2893, 2746, 2611, 2421, 1614, 1583, 1530, 1457,
1436, 1304, 1252, 1181, 1102, 1029, 982, 933, 824, 746; ; MS (ESI) m/z [M-H]- 367.9(HCl salt) ;
Anal. Calcd. for C20H20ClN3O2; C, 64.95; H, 5.45; Cl, 9.59; N, 11.36; O, 8.65 %. Found C, 65.11; H,
5.35; N, 11.46 %.
5.1.3.8. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-methoxyphenyl)isoxazol-5-yl)methanamine
hydrochloride (10h)
1
Yield: 73 %; off white solid; mp 202 – 204 ˚C; H NMR (400 MHz, DMSO-d6) δ: 3.87 (s,
3H), 4.44 (d, 4H), 7.07 (d, 2H, J = 4.8 Hz) 7.18 (s, 1H), 7.26 (dd, 1H, J = 2.0 Hz, J = 8.4 Hz), 7.41
13

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(d, 1H, J = 8.8 Hz), 7.66 (d, 1H, J = 4.0 Hz), 7.81 (d, 2H, J = 4.0 Hz), 8.04 ( d, 1H, J = 4.0 Hz),
9.79 (d, 1H), 11.66 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ:40.2, 41.6, 55.8, 104.2, 104.6, 112.4,
114.2, 115.0 (2C), 120.8, 121.8, 124.5, 128.5 (2C), 129.0, 129.9, 135.1, 161.3, 162.1, 164.9; IR
(KBr) cm-1 : 3208, 3001, 2939, 2743, 2565, 2409, 1613, 1564, 1532, 1432, 1394, 1299, 1259, 1178,
1108, 1030, 979, 948, 904, 886, 838, 802; MS (ESI) m/z [M-H]- 410.5 ; Anal. Calcd. for
C20H19BrClN3O2; C, 53.53; H, 4.27; Br, 17.81; Cl, 7.90; N, 9.36; O, 7.13 %. Found C, 53.73; H,
4.01; N, 9.76 %.
5.1.3.9. (3-(4-methoxyphenyl)isoxazol-5-yl)-N-((5-methyl-1H-indol-3-yl)methyl)methanamine
hydrochloride (10i)
1
Yield: 65 %; off white solid; mp 177 – 179 ˚C; H NMR (400 MHz, DMSO-d6) δ: 2.39 (s,
3H), 3.82 (s, 3H), 4.41(d, 4H), 6.98 (d, 1H, J = 8.0 Hz), 7.08 (d, 2H, J = 8.8 Hz), 7.18 (s, 1H),
7.32 ( s, 1H, J = 8.4 Hz), 7.55 – 7.53 (m, 2H), 7.80 (d, 2H, J = 8.8 Hz), 9.75 (s, 2H), 11.31 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 55.8, 104.1, 104.2, 111.9, 115.0, 118.6, 120.8,
123.6, 124.6, 127.4, 128.1, 128.3, 128.5 (2C), 134.7, 161.3, 162.1, 164.9; IR (KBr) cm-1 : 3125,
3080, 2836, 2709, 1699, 1615, 1576, 1529, 1469, 1387, 1350, 1283, 1257, 1175, 1090, 1020, 919,
900, 837, 808; MS (ESI) m/z [M-H]- 381.9 (HCl salt); Anal. Calcd. for C21H22ClN3O2 C, 65.71; H,
5.78; Cl, 9.24; N, 10.95; O, 8.34 %. Found C, 65.91; H, 5.58; N, 10.97 %.
5.1.3.10. N-((1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanaminehydrochloride
(10j)
1
Yield: 70 %; off white solid; mp 212 – 214 ˚C 0C;; H NMR (400 MHz, DMSO-d6) δ:
4.48 (d, 4H, J = 28 Hz), 7.18 – 7.08 (m, 2H), 7.39 (s, 1H), 7.44 (d, 1H, J = 8.0 Hz), 7.60 ( d, 1H, J
= 4.0 Hz),7.77 (d, 1H, J = 8.0 Hz), 8.17 (d, 2H, J = 4.0 Hz), 8.39 ( d, 2H, J = 8.0Hz), 9.77 ( s, 2H),
14

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13
11.43 (s, 1H); C NMR (100 MHz, DMSO-d6) δ: 40.5, 42.03, 104.8, 104.9, 112.2, 119.1, 119.6,
122.1, 124.9 (2C), 127.1, 128.2, 128.4 (2C), 134.4, 136.4, 148.9, 161.2, 166.31; IR (KBr) cm-1 :
3440, 3125, 2946, 2780, 2602, 2422, 1695, 1600, 1522, 1462, 1433, 1347,1290, 1170, 1103, 1015,
938, 833, 858, 754; MS (ESI) m/z [M-H]- 383.2 (HCl salt) ; Anal. Calcd. for C19H17ClN4O3; C,
59.30; H, 4.45; Cl, 9.21; N, 14.56; O, 12.47 %. Found C, 59.21; H, 4.35; N, 12.36 %.
5.1.3.11. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanamine
hydrochloride (10k)
1
Yield: 79%; off white solid; mp 188 – 190 ˚C; H NMR (400 MHz, DMSO-d6) δ: 4.47 (d,
4H, J = 24 Hz), 7.25 (dd, 1H, J = 1.6 Hz, J = 8.8 Hz), 7.43 – 7.40 (m, 2H), 7.69 ( d, 1H, J = 2.0 Hz),
13
8.04 (s, 1H), 8.15 (d, 2H, J = 8.0 Hz), 8.39 (d, 2H, J = 8.0 Hz), 10.01 (s, 2H), 11.70 (s, 1H); C
NMR (100 MHz, DMSO-d6) δ: 40.5, 41.7, 104.6, 104.9, 112.4, 114.2, 121.8, 124.5, 124.9 (2C),
128.4 (2C), 129.0, 129.9, 134.4, 135.1, 161.1, 166.2 (2C); IR (KBr) cm-1 : 3318, 3099, 2943, 2722,
1601, 1522, 1451, 1347, 1290, 1231, 1169, 1111, 1045, 984, 923, 886, 855, 806, 755, 698; MS
(ESI) m/z [M-H]- 425.5 ; Anal. Calcd. C19H16BrClN4O3;C, 49.21; H, 3.48; Br, 17.23; Cl, 7.65; N,
12.08; O, 10.35 %.Found C, 49.39; H, 3.23; N, 12.01 %.
5.1.3.12. N-((5-methyl-1H-indol-3-yl)methyl)(3-(4-nitrophenyl)isoxazol-5-yl)methanamine
hydrochloride (10l)
Yield: 70 % ;off white solid; mp 198 – 200˚C; 1H NMR (400 MHz, DMSO-d6) δ: 2.40 (s,
3H), 4.45 (d, 4H, J = 37.2 Hz), 6.98 (d, 1H, J = 8.0 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.39 (s, 1H), 7.54
13
(s, 2H), 8.16 (d, 2H, J = 8.0 Hz), 8.39 (d, 2H, J = 8.0 Hz), 9.70 (s, 2H), 11.29 (s, 1H); C NMR
(100 MHz, DMSO-d6) δ: 21.7, 40.3, 40.6, 104.1, 104.9, 118.6, 123.7, 124.9 (2C), 127.3, 128.1,
128.27, 128.4 (2C), 134.4, 134.7, 148.9, 161.6 , 166.2 (2C).;IR (KBr) cm-1 :3399, 3117, 2776, 1703,
15

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1598, 1556, 1526, 1431, 1349, 1291, 1247, 1217, 1169, 1089, 1039, 1016, 975, 856, 836, 808, 758,
714, 699; MS (ESI) m/z [M-H]- 397.2 (HCl salt) ; Anal. Calcd. for C20H19ClN4O3;C, 60.23; H, 4.80;
Cl, 8.89; N, 14.05; O, 12.03%.Found C, 60.43; H, 4.70; N, 14.34 %.
5.1.3.13. N-((1H-indol-3-yl)methyl)(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5-
yl)methanamine hydrochloride (10m)
Yield: 70 %; off white solid; mp 165 - 167 ˚C; 1HNMR (400 MHz, DMSO-d6) δ: 4.46 (d,
4H, J = 18.8 Hz), 7.09 (t, 1H, J = 4.0Hz), 7.16 (t, 1H, J = 8.0Hz), 7.43 ( t, 2H, J = 8.0Hz), 7.63 (d,
1H, J = 4.0Hz), 7.78 – 7.71 (m, 2H), 8.20 (dd, 1H, J = 4.0Hz, J = 8.0Hz), 8.28 – 8.25 (m, 1H), 9.92
(s, 2H), 11.45 (s, 1H);13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.8, 104.6, 104.8, 112.2, 117.9,
118.1, 118.9, 119.1, 119.6, 121.3, 122.0, 124.0, 125.7, 127.2, 128.3, 134.1, 136.4, 160.7, 165.9; IR
(KBr) cm-1 : 3442, 3127, 2890, 2782, 2610, 2421, 1614, 1522, 1457, 1430, 1384, 1325, 1272, 1245,
1140, 1105, 1080, 1053, 1015, 968, 937, 907, 833, 750; MS (ESI) m/z [M-H]- 424.4 (HCl salt) ;
Anal. Calcd. for C20H16ClF4N3O; C, 56.41; H, 3.79; Cl, 8.33; F, 17.85; N, 9.87; O, 3.76%.Found C,
56.66; H, 3.98; N, 9.66%.
5.1.3.14. N-((5-bromo-1H-indol-3-yl)methyl)(3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5-
yl)methanamine hydrochloride (10n)
Yield: 73%; off white solid; mp 176 – 178 ˚C;1H NMR (400 MHz, DMSO-d6) δ: 4.47 (d, 4H,
J = 23.6 Hz), 7.26 (d,1H, J = 8.0Hz), 7.35 – 7.45 ( d, 2H, J = 4.0Hz), 7.65 ( d, 1H, J = 4.0Hz), 7.75
(t, 1H, J = 8.0Hz), 8.02 ( d, 1H, , J = 4.0Hz), 8.20, (d, 1H, J = 4.0Hz), 8.29 – 8.25 (m, 1H), 9.80 (s,
2H), 11.64 (s, 1H);13C NMR (100 MHz, DMSO-d6) δ: 40.5, 41.6, 104.6, 104.8, 112.4, 114.2, 118.0,
118.9, 119.1, 121.3, 121.8, 124.0, 124.5, 125.7, 129.0, 130.0, 134.0, 135.1, 160.7, 165.9;IR (KBr)
cm-1 : 3347, 3111, 2973, 2732, 2606, 2455, 1611, 1524, 1458, 1431, 1400, 1377, 1321, 1270, 1249,
16

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1170, 1132, 1081, 1053, 968, 945, 905, 871, 835, 798; MS (ESI) m/z [M-H]- 504.2 (HCl salt);Anal.
Calcd. for C20H15BrClF4N3O; C, 47.60; H, 3.00; Br, 15.83; Cl, 7.02; F, 15.06; N, 8.33; O,
3.17 %.Found C, 47.89; H, 2.71; N, 8,23 %.
5.1.3.15. (3-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazol-5-yl)-N-((5-methyl-1H-indol-3-
yl)methyl)methanamine (10o)
1
Yield: 70%; off white solid; mp 155 -156 ˚C; H NMR (400 MHz, DMSO-d6) δ: 2.39 (s,
3H), 4.40 (s, 2H), 4.48 (s, 2H), 6.98 (d, 1H, J = 8.0 Hz), 7.32 ( d, 1H, J = 8.0 Hz), 7.40 ( s, 1H),
7.50 – 7.60 (m , 2H), 7.75 ( t, 1H, J = 4.0 Hz), 8.21, (d, 1H, J = 8.0 Hz), 8.27 (m, 1H), 9.75 (s, 2H),
11.31 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ: 21.7, 40.5, 41.8, 104.1, 104.8, 111.9, 118.6, 118.9,
119.1,121.5, 123.7,124.5, 125.7, 127.4, 128.1, 128.3, 130.0, 134.1, 134.8, 160.73, 165.9; IR (KBr)
cm-1 : 3347, 2949, 2778, 2592, 1742, 1693, 1626, 1523, 1429, 1400, 1321, 1266, 1168, 1134, 1079,
1054, 833, 771, 704, 679; MS (ESI) m/z [M-H]- 438.3 (HCl salt);Anal. Calcd. for C21H18ClF4N3O;
C, 57.35; H, 4.12; Cl, 8.06; F, 17.28; N, 9.55; O, 3.64% Found C, 57.50; H, 4.42; N, 9.35 %.
5.2.
Pharmacology
5.2.1. Chemicals and reagents
Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS), pencillin and
streptomycin were purchased from Gibco BRL (Life Technologies) Rockville, MD, USA). MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], doxorubicin, λ-carrageenan and
ursolic acid were purchased from Sigma-Aldrich Chemical Company, (St. Louis, MO, USA).
5.2.2. In vitro secretory phospholipase A2 assay
17

ACCEPTED MANUSCRIPT
sPLA2 assay was performed using type IIA sPLA2 enzyme activity kit (Cayman Chemical,
Ann Arbor, MI, USA) according to the manufacturer’s instructions. The reaction mixture contained
10 µl of sPLA2, 10 µl of inhibitor (test wells only), 200 µl substrate and was incubated for 15 min.
Then, 10 µl of 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) was added to develop the colour and
read at a wavelength of 415 nm. sPLA2 enzyme hydrolysed the thioester bond at the sn-2 position of
diheptanoyl thio-PC (substrate), free thiols were released and reacted with DTNB to form 5-thio-2-
nitrobenzoic acid, which had absorbance at 415 nm. The control wells contained only sPLA2,
substrate and DTNB. Urosolic acid was used as a positive control [19]. The percent inhibition of
enzyme activity was calculated using the following formula:
% inhibition = [(Absorbance of control – Absorbance of test/Absorbance of control) ×100].
5.2.3. In vivo anti-inflammatory activity by carrageenan-induced rat paw edema model
Carrageenan-induced paw edema model is the most widely used for the evaluation of anti-
inflammatory activity [12]. Male wistar albino rats weighting 150 - 200 g were obtained from M/S
Mahavir Enterprises (Hyderabad, Telangana, India). The animals were housed under standard
conditions (temperature of 22±100C with an alternating 12-h light-dark cycle and relative humidity
of 60±5%, before and also during the experiment. The animals were fed with standard laboratory
diet,
which
was
purchased
from
M/S
Rayans
Biotechnology Pvt.
Ltd.
(Hyderabad, Telangana, India). During the experiment, the rats were allowed water and food ad
libitum. Animal experimental protocol was approved by institutional animal ethical committee
(IAEC no. GU/GIS/IAEC/PROTOCOL NO.03/2013). Animal experiments were conducted
according to CPCSEA guidelines.
The animals were divided into three groups (n = 3-6). Group I served as control and received
saline at 1 ml/kg body weight (b.w) per os (p.o). Group II served as standard, received ursolic acid
18

ACCEPTED MANUSCRIPT
at a dose of 5 mg/kg b.w, p.o. and group III served as test and received synthesized compound(s) at
a dose of 5 mg/kg b.w, p.o, respectively.
The animals were treated with synthesized compounds and ursolic acid before 1 h to inject
(subcutaneous) 0.1 ml of 1% λ-carrageenan solution with saline into sub-plantar region of left hind
paw of each rat. The right hind paw of same rat was treated with 0.1 ml of saline alone in manner as
control. Before induction of edema, the dorsiventral thickness of both the paws of each was
measured using mercury displaced glass plethysmometer and also the measurements were taken at
1st, 2nd, 3rd, 4th and 6th hour after carrageenan injection. The results are expressed in percent edema
inhibition. Ursolic acid was used as a positive control.
5.2.4. Anticancer studies
5.2.4.1. Cancer cell lines
MCF-7 breast cancer cell line was purchased from National Cancer Institute (Frederick,
MD, USA). Human prostate carcinoma cell line DU145 was obtained from American Type Culture
Collection (Manassas, VA, USA). These cancer cells were maintained in RPMI medium
supplemented with 10% FCS, 100 units pencillin and 100 µg of streptomycin per ml of medium.
The cells were maintained in 95% humidity with CO2 at 37 0C temperature.
5.2.4.2. Cell proliferation by MTT assay
MTT assay was performed to assess the antiproliferative activity of compounds [20]. Cancer
cells were treated with isolated compound at various concentrations for 48 h. 20 µl of freshly
prepared MTT reagent was added to cells and incubated at 37 °C for 2 h. The supernatant growth
medium was removed and replaced with DMSO (100 µl) to dissolve the formazan crystals. The
optical density (absorbance) was read at 570 nm with reference wavelength of 620 nm.
5.2.5. In silico studies
19

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5.2.5.1. Drug like properties
The rule was formulated by Christopher A. Lipinski in 1997 [21]. The rule describes the
molecular properties important for a drug’s pharmacokinetics in the human body, including their
absorption, distribution, metabolism and excretion (ADME). The rule is important for drug
development where a pharmacologically active lead structure is optimized stepwise for increased
activity and selectivity, as well as druglike properties. Molinspiration tool used to demonstrate the
druglike properties of compounds [22].
5.2.5.2. Molecular docking studies
X-ray crystal structures of human group IIA sPLA2 (PDB ID: 1DB5) protein was obtained
from Protein Data Bank and used in docking studies. Co-cystalized ligands and water molecules
were removed from target protein using Argus lab. Ligands are prepared using chemoffice
(Cambridge, UK). Energy minimization was done using molecular mechanics. The minimization
was executed until root mean square value reached below 0.001 Kcal/mol. Such energy minimized
ligands and receptors were used for docking studies using GEMDOCK (Generic Evolutionary
Method for molecular DOCKing), which is a generic evolutionary method with an empirical
scoring function for molecular docking (protein–ligand docking) [12, 23, 24]. A population size of
300 with 70 generations and 3 solutions were used in docking accuracy setting. PyMol was used for
better visualization of interactions [23-25].
Conflict of Interest: The authors declare no conflict of interest.
Acknowledgements
SRP and GR are grateful to Sarada Villas College Mysore for the support and encouragement to
higher education. SRP and GR also thankful to CRD Department of Chemistry, PRIST University,
20

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Thanjavur-613403, India. NSY are thankful to GITAM University for providing research facilities.
NSY expresses his sincere gratitude to Dr. M.V.V.S. Murthy, Pesident, GITAM University for his
support and encouragement by providing necessary research facilities. NSY is very much thankful
to Prof. G. Subrahmanyam, Prof. N. Lakshmana Das and Prof. Ch. Ramakrishna for their
encouragement and motivation. This work was partly supported by the Russian Sciene Foundation
(www.rscf.ru, Российский Научный Фонд, grant number 14-23-00160 for 2014-2016).
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Legends to figures:
Figure 1. In vitro sPLA2 inhibitory activity of 10o and Ursolic acid (positive control). Compound
10o dose-dependley inhibited sPLA2 activity with IC50 of 10.23±0.91 ꢀM where as ursolic acid
(positive control) inhibited with IC50 of 12.59±1.03 ꢀM.
Figure 2. Molecular docking studies of 10o on human GIIA sPLA2 crystal protein. (A) Binding
orientation of 10o on crystal structure of human GIIA PLA2 (PDB ID: 1DB5) in ribbon model. (B).
10o binds vicinity of amino acid residues at active site were PHE-5, TYR-21, GLY-22, CYS-28,
GLY-29, CYS-44, HIS-47, ASP-48, TYR-51, PHE-98.
Figure 3. Anti-inflammatory activity of 10o. (A) Represents the rat paw edema volumes (ml) of
treated (10o and ursolic acid) and untreated at various time intervals (1, 2, 3, 4 and 6 hrs) after
carrageenan injection. Experimental results are expressed as mean (n = 3-6) ± standard error mean
(S.E.M). The difference between experimental groups was compared by one-way analysis of
variance (ANOVA). The P value of results less than 0.05 (P˂0.05) versus control was considered as
significant (*represents p<0.05, **p<0.001). (B). 10o showed comparable activity with ursolic acid
(positive control) and exhibited maximum edema inhibition between 3rd and 4th h after carrageena
injection.
Figure 4. Antiproliferative effect of 10o against MCF-7 breast and DU145 prostate cancer cells.
Compound 10o showed significant anti-proliferative effect in MCF-7 breast and DU145 prostate
cancer cell lines with IC50 of 10.03±0.86 and 14.15±1.2 µM, respectively, whereas doxorubicin
(positive control) exhibited anticancer activity with 8.02±0.69 and 4.75±0.35 µM, respectively.
23

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Br
O
N
N3
OH
N
O
N
O
H
iii
i
ii
R2
R2
R2
R2
R1
R1
R1
3
R1
2
1
4
NH2.HCl
O
N
iv
5e=R1=F,R2=CF3
5b=R1=CH3,R2=H,
5a=R1=H,R2=H,
R2
5d=R1=NO2,R2=H,
5c=R1=OCH3,R2=H,
R1
5(a-e)
O
R
O
R
H
8a=R=H,
8b=R=Br,
R
H
vi
v
N
N
Boc
N
H
H
8a=R=CH3
6
7
8(a-c)
NH2.HCl
O
O
N
N
O
R
R
HN
H
vii
R1
N
Boc
R2
N
Boc
R2
R1
5(a-e)
9(a-o)
8(a-c)
viii
10b=R=Br,R1=H,R2=H,
10a=R=H,R1=H,R2=H,
10c=R=CH3,R1=H,R2=H,
10d=R=H,R1=CH3,R2=H,
10f=R=CH3,R1=CH3,R2=H,
O
N
10e=R=Br,R1=CH3,R2=H,
R
HN
.HCl
10h=R=Br,R1=OCH3,R2=H,
10j=R=H,R1=NO2,R2=H,
10g=R=H,R1=OCH3,R2=H,
10i=R=CH3,R1=OCH3,R2=H,
10k=R=Br,R1=NO2,R2=H,
10m=R=H,R1=F,R2=CF3,
R1
N
R2
H
10(a-o)
10l=R=CH3,R1=NO2,R2=H,
10n=R=Br,R1=F,R2=CF3,
10o=R=CH3,R1=F,R2=CF3,
(i) NH2OH.HCl, NaOH, H2O, rt, (ii) Chloramino-T, Propargyl bromide, EtOH, 60-65 °C, (iii) NaN3, DMF,
60-65 °C, (iv) PPh3, H2O, THF, rt v) POCl3, DMF, rt, (Vi) NaH, Boc anhydride, DMF, rt. (Vii) NaCNBH3,
MeOH (Viii) EtOAc in HCl
Scheme 1: Synthesis of indole containing isoxazole derivatives (10 a-o)
24

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Table 1
GIIA sPLA2 inhibitory activities of indole containing isoxazole derivatives.
Compound no
In silico analysis
In vitro
IC50 ( M)
µ
Dock score
(-Kcal/mol)
-107.3
30.63±0.91
32.49±1.02
27.92±0.86
22.68±1.13
27.32±1.73
21.55±1.21
18.22±1.42
32.64±2.38
24.55±2.81
25.61±1.72
22.35±2.49
19.44±2.1
10a
10b
10c
10d
10e
10f
-109.6
-111.8
-115.4
-118.4
-111.3
-112.3
-103.8
-105.6
-108.2
-103.7
-104.1
-108.7
-110.3
-129.5
-128.8
10g
10h
10i
10j
10k
10l
21.5±1.52
10m
10n
10o
17.18±1.43
10.23±0.91
12.59±1.03
Ursolic acid
(positive control)

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Table 2
Drug like properties of indole containing isoxazole derivatives.
Compound
10a
MW
Log P
3.442
4.227
3.866
3.89
Non
4
NoHnH
303.365
382.261
317.392
317.392
396.28
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
10b
10c
4
4
10d
10e
4.675
4.325
3.498
4.283
3.923
3.4
4
331.41
4
10f
333.39
5
10g
412.287
347.418
348.362
427.258
362.389
389.352
468.248
403.379
5
10h
10i
5
7
10j
4.186
3.825
4.429
5.214
4.853
7
10k
10l
7
4
10m
10n
10o
4
4

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