Screening of quinoline, 1,3-benzoxazine, and 1,3-oxazine-based small molecules against isolated methionyl-tRNA synthetase and A549 and HCT116 cancer cells including an in silico binding mode analysis

Article abstract of DOI:10.1039/c5ob00791g

Elevated activity of methionyl-tRNA synthetase (MRS) in many cancers renders it a possible drug target in this disease area, as well as in a series of parasitic diseases. In the present work, we report the synthesis and in vitro screening of a library of 1,3-oxazines, benzoxazines and quinoline scaffolds against human MRS. Among the compounds tested, 2-(2-butyl-4-chloro-1-(4-phenoxybenzyl)-1H-imidazol-5-yl)-5-(4-methoxyphenyl)-1-oxa-3-azaspiro[5.5]undecane (compound 21) and 2-(2-butyl-4-chloro-1-(4-nitrobenzyl)-1H-imidazol-5-yl)-2,4-dihydro-1H-benzo[d][1,3]oxazine (compound 8) were found to be potent inhibitors of MRS. Additionally, these compounds significantly suppressed the proliferation of A549 and HCT116 cells with IC₅₀ values of 28.4, 17.7, 41.9, and 19.8 μM respectively. Molecular docking studies suggested that the ligand binding orientation overlaps with the original positions of both methionine and adenosine of MRS. This suggests the binding of compound 21 against MRS, which might lead the inhibitory activity towards cancer cells.

Full text of DOI:10.1039/c5ob00791g

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Mohan, S. Rangappa, T. Kang, K. H. Kumar, J. Fuchs, K. N. Hoon, A. Bender, S. Kim, S. Basappa and K. S.
Rangappa, Org. Biomol. Chem., 2015, DOI: 10.1039/C5OB00791G.
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Page 1 of 23
Organic & Biomolecular Chemistry
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DOI: 10.1039/C5OB00791G
Screening of Quinoline, 1,3ꢀBenzoxazine, and 1,3ꢀOxazineꢀbased Small Molecules Against
Isolated MethionylꢀtRNA Synthetase and A549 and HCT116 Cancer Cells Including an In
Silico Binding Mode Analysis
a,#
b,#
c
Hanumantharayappa Bharathkumar, Chakrabhavi Dhananjaya Mohan, Shobith Rangappa,
d
a
e
d
e
Taehee Kang, Keerthy H K, Julian E. Fuchs, Nam Hoon Kwon, Andreas Bender, Sunghoon
d,f
a,*
b,*
Kim, Basappa, Kanchugarakoppal S Rangappa,
a
Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Palace
Road, Bangalore 560 001, India
b
Department of Studies in Chemistry, University of Mysore, Mysore 570 006, India
c
Frontier Research Center for Post-genome Science and Technology Hokkaido University, Japan
d
Medicinal Bioconvergence Research Center, Seoul National University, Seoul 151-742, Korea
e
Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, Lensfield
Road, CB2 1EW, Cambridge, United Kingdom
f
WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of
Convergence Science and Technology, Seoul National University, Suwon 443-270, Korea.
#
Contributed Equally
1

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Abstract
Elevated activity of methionylꢀtRNA synthetase (MRS) in many cancers renders it a
possible drug target in this disease area, as well as in a series of parasitic diseases. In the present
work, we report the synthesis and in vitro screening of a library of 1,3ꢀoxazines, benzoxazines
and quinoline scaffolds against human MRS. Among the compounds tested, 2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ
1ꢀ(4ꢀphenoxybenzyl)ꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecane
(compound 21) and 2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀnitrobenzyl)ꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀ
benzo[d][1,3]oxazine (Compound 8) were found to be potent inhibitors of MRS. Additionally,
those compounds significantly suppressed the proliferation of A549 and HCT116 cells with an
IC value of 28.4, 17.7 and 41.9, 19.8 µM respectively. Molecular docking studies suggested
5
0
that the ligand binding orientation overlaps with the original positions of both methionine and
adenosine of MRS. This suggests the binding of compound 21 against MRS, which might leads
the inhibitory activity towards cancer cells.
2

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Organic & Biomolecular Chemistry
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Introduction
AminoacylꢀtRNA synthetases (ARS) are a group of enzymes that transfer a specific
1
amino acid to cognate tRNA to form aminoacylꢀtRNA. The amino acylatedꢀtRNA then carries
the respective amino acid to the site of protein synthesis to begin translation at the initiation
2
codon (or to continue this process). The initiation codon codes for methionine, which is hence
the first amino acid added during protein synthesis. Methionine specifically is esterified to
met
tRNA by methionylꢀtRNA synthetase (MRS), and elevated activity of MRS is reported in
3
ꢀ5
human cancers. In cancer cells, targeting translational initiation is considered as one of the
6
effective strategy to inhibit the cell survival, proliferation and metastasis. Selective inhibition of
human MRS hence results in blockade of early events of protein synthesis, and thereby
7
termination of global translation. The human MRS upꢀregulation in cancer renders it as a unique
target to design inhibitors, thereby inhibiting its functional properties.
Research in the previous decades explored numerous scaffolds including quinolines,
pyrrolines and chloramphenicol derivatives as inhibitors of bacterial MRS, and most of them
8
, 9
displayed high specificity towards bacterial MRS and failed in inhibiting human MRS.
Quinoline derivatives in particular have been extensively presented as inhibitors of bacterial
MRS, and more recently the quinolineꢀ4ꢀone based small molecule REP8839 was reported as a
novel inhibitor of MRS in methicillinꢀresistant Staphylococcus aureus as well as Streptococcus
1
0
pyogenes. . Furthermore, benzoxaborole derivatives were shown to have significant leucylꢀ
1
1
tRNA inhibitory activity. Overall, these studies indicate that many quinolineꢀ, oxazineꢀ and
benzoxazineꢀbased small molecules could be good candidates for the development of potent
inhibitors against human as well as bacterial MRS.
3

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We previously reported novel synthetic routes for the preparation of quinoline,
1
2ꢀ15
benzoxazine and oxazine scaffolds and their derivatives.
Herein, we report the design,
synthesis, and biological evaluation of quinolines, benzoxazines, and oxazine derivatives against
human methionylꢀtRNA synthetase as well as cancer cell lines, including an analysis of putative
binding modes against the target enzyme.
Results and Discussion
Chemistry
Since oxazineꢀbased compounds have been reported to have good antioncogenic activity in
hepatocellular carcinoma and osteosarcoma model, we here attempted to prepare newer oxazines
by reacting 1ꢀ(2ꢀaminoꢀ1ꢀ(4ꢀmethoxyphenyl)ꢀethyl)ꢀcyclohexanol with various aldehydes in the
presence of potassium carbonate in methanol media (Scheme 1). Further, 1,3ꢀbenzoxazines were
prepared by reacting 2ꢀamino benzyl alcohol with different aldehydes in the presence of
chloroacetic acid in methanolic media (Scheme 2). Quinolines were prepared using 2ꢀamino
benzyl alcohol with various aldehydes in T3P (propylphosphonic anhydride) catalyzed reaction
using Microwave Irradiation (Monowave G10 apparatus) (Scheme 3).The compounds obtained
1
13
were characterized by melting point, H NMR, C NMR, and mass spectral analysis. Detailed
chemical characterizations of the newly synthesized compounds are provided in the Materials
and Methods section.
In vitro screening of the library of small molecules against human MRS
Initially all the synthesized compounds were evaluated for their in vitro inhibitory
activity against human MRS at the concentration of 100 µM. Among the newly synthesized
structures, compounds 8, 19, 21, 23, 26 and 29 reduced aminoacylation activity of MRS over 60
%
while compound 1 displayed minimal or no inhibitory activity (see Figure 1) and the MRS
4

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activity was expressed based on the radioactivity (CPM: count per minute) of 35[S]ꢀMet charged
tRNA. Methionine analog, FmocꢀDLꢀselenomethionine (Anaspec), was used as a positive
control. The lead compounds were further evaluated at 25 µM and 100 µM and the results are
expressed as relative inhibitory activity (see Figure 2). The relative inhibitory activity was
calculated by converting CPM values of compound treated reaction into percentage inhibition
with that of negative control (100 %). All the shortlisted compounds displayed a substantial
decrease of MRS activity in a doseꢀdependent manner and compound 21, which bears 1,3ꢀ
oxazine ring attached to the substituted imidazole moiety was identified as the most potent
inhibitor of human MRS. Further, we observed that, attachment of substituted imidazole moiety
to oxazines and benzoxazines, haloarenes to quinolines contributed to the inhibition of MRS
catalytic activity.
Compound 21 and compound 8 suppress the proliferation of A549 and HCT116 cancer cell
lines
1
6, 17
Given the activity of MRS in various cancers,
we next investigated the
antiproliferative potential of the six lead compounds against A549 (lung carcinoma) and
1
8
HCT116 (colon cancer) cells using the MTT assay. Paclitaxel was used as the reference drug.
Among the tested compounds, compound 21 was found to be the most effective antiproliferative
agent with the IC values of 28.4 and 17.7 µM, respectively, against the A549 and HCT116 cell
5
0
lines, followed by compound 8 with IC values of 41.9 and 19.8 µM (see Table 1). The results
5
0
of the cytotoxicity readout are hence closely correlated with in vitro MRS enzyme activity.
Molecular docking studies
Computational docking studies were performed next to understand the molecular
interactions between the bioactive small molecules synthesized and tested earlier using
5

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methionylꢀtRNA synthetase (MRS) as the target structure (see Materials and Methods for
details). We found consistent predicted binding modes for the oxazine series, placing the
protonated nitrogen of the oxazines in vicinity of Asp296, thus forming chargeꢀassisted hydrogen
bonds and occupying the ribose position of the coꢀcrystallized ligands. Furthermore, docked
ligands were found to show major volume overlap with the original positions of both methionine
and adenosine (Figure 3). Compound 21, which has been found to be most active in vitro as well
as exhibiting highest docking scores, is predicted to form πꢀπ interactions via a methoxy phenyl
group with Tyr15 and Trp253, that are also key anchor points for the substrate, methionine. A
chloroimidazole moiety replaces the adenine base and a hydrophobic substituent forms
additional πꢀstacking with His24, thus providing a hypothesis for the activity identified for this
structure.
Experimental
Chemicals
All chemicals used were of analytical grade and purchased from Sigma Aldrich, and SRL,
Mumbai (India). All IR spectra were obtained in a KBr disc on a Shimadzu FTꢀIR 157
1
Spectrometer. H NMR spectra were recorded on a Bruker (400 MHz) spectrometer in CDCl or
3
1
3
DMSOꢀd6 as solvent, using TMS as an internal standard, C NMR spectra were recorded on a
Bruker/Agilent (100 MHz) spectrometer and chemical shifts were expressed as δ ppm and
abbreviations are assigned as, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet and J values
are given in Hz. Mass spectra were determined on a Shimadzu LCꢀMS and ESIꢀMS, Elemental
analyses were carried out using an Elemental Vario Cube CHNS Rapid Analyzer. Progress of the
reaction was monitored by TLC preꢀcoated silica gel plates.
6

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General procedure for the synthesis of 1,3ꢀoxazines
(1ꢀ7,9, 12ꢀ13 & 21): Synthesis of 1ꢀ(2ꢀ
amino)ꢀ1ꢀ(4ꢀmethoxyphenylethyl) cyclohexanol (AA) - We initially prepared 1ꢀ[2ꢀaminoꢀ1ꢀ(4ꢀ
1
5
methoxyꢀphenyl)ꢀethyl]ꢀcyclohexanolmonoacetate as described earlier and briefly summarized
here. To stirred solution of 1ꢀ(2ꢀamino)ꢀ1ꢀ(4ꢀmethoxyꢀphenylꢀethyl)ꢀ
a
cyclohexanolmonoacetate(1 eq) in methanol (10 mL) aldehydes (1 eq) and anhydrous potassium
carbonate (2.5 eq) were added and the reaction mixture was stirred at room temperature for 9 h.
After completion of the reaction, methanol was evaporated and water was added and extracted
with ethyl acetate (15 mL). The combined organic layer was dried over anhydrous sodium
sulphate. The crude solid was obtained on evaporation of the solvent under reduced pressure and
recrystallized from hexane and ethyl acetate to furnish crystalline solid (Scheme 1)
Synthesis of 2ꢀ(2,6ꢀdifluorophenyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecane (1)
1
5, 19
was carried out using the reported protocol.
Synthesis of 2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀ
azaspiro[5.5]undecane (2): Compound 2 was obtained from AA (1 mmol), 2ꢀbutylꢀ4ꢀchloroꢀ
Hꢀimidazoleꢀ5ꢀcarbaldehyde (1 mmol) and K CO (2.5 mmol) as reddish brown crystalline
1
2
3
0
solid, Yield: 89%, Melting point:108ꢀ110 C; Elemental Analysis Calculated for C H ClN O :
2
3
32
3
2
ꢀ1
C, 66.09; H, 7.72; N, 10.05; found C, 66.14; H, 7.51; N, 10.13%; IR νmax (KBr, cm ): 3340,
1
2
895, 1050; H NMR ( CDCl 400 MHz ) δ: 7.85 (s, 1H), 7.15 (d, 2H), 6.8 (d, 2H), 4.9 (s, 1H),
3
,
1
3
4
.2 (s,1H), 3.9 (m, 1H), 3.75 (s, 3H), 2.9 (dd, 2H), 2.6 (t, 2H), 1.8 ꢀ1.2 (m, 14H), 0.9 (t,3H); C
NMR (DMSO, 100 MHz)δ: 157.09, 147.73, 135.89, 134.55, 129.56, 120.81, 115.03, 88.62,
9.84, 55.60, 51.87, 48.12, 37.10, 36.93, 31.01, 28.20, 27.04, 22.97, 21.87, 20.95, 14.24; Mass:
m/z found for C H ClN O : 418.2, 419.2.
7
23
32
3
2
7

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Synthesis of 2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecane (3):
Compound 3 was obtained from AA (1 mmol), indoleꢀ3ꢀcarbaldehyde (1 mmol) and K CO3
2
0
(
2.5mmol) as brown crystalline solid, Yield: 90%; Melting point: 112ꢀ113 C; Elemental Analysis
Calculated for C H N O : C, 76.56; H, 7.50; N, 07.44; found C, 76.65; H, 7.51; N, 07.23%; IR
2
4
28
2
2
ꢀ1
1
νmax (KBr, cm ): 3260, 2910,1150, H NMR (DMSO 400 MHz) δ: 11.5 (s, 1H), 8.07ꢀ8.05 (d,
,
J=7.6 Hz, 1H) 7.71 (s, 1H), 7.45ꢀ7.43(d, J=8Hz, 1H), 7.24ꢀ7.17 (dd,J =8Hz, J = 8Hz,4H),7.08ꢀ
1
2
7
3
1
8
.11(t, J=7.6,1H) 6.8 (d, J=8.0Hz, 1H), 4.85(s,1H), 4.22(s,1H), 3.84ꢀ3.81(m, 1H), 3.73(s,3H),
1
3
.0ꢀ2.98(d, J=5.6,1H), 1.71ꢀ1.11(m,10H), C NMR (DMSO, 100 MHz )δ: 157.56, 136.92,
33.13, 130.93, 130.53, 130.41, 124.79, 122.32, 121.29, 120.29, 114.25, 113.12, 112.85, 111.71,
9.25, 72.28, 55.87, 54.75, 41.58, 36.92, 34.10, 25.58, 21.61, 21.33; Mass: m/z found for
+
C H N O : 377.4 ([M+1] )
24
28
2
2
Synthesis of 2ꢀ(4ꢀbromophenyl)ꢀ5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecane (4):
Compound 4 was obtained from AA (1 mmol), 4ꢀBromo benzaldehyde (1 mmol) and K CO3
2
0
(
2.5 mmol) as colorless crystalline solid, Yield: 90%; Melting point: 79ꢀ80 C; Elemental
Analysis Calculated for C H BrNO : C, 63.46; H, 6.29; N, 03.36; found C, 63.34; H, 6.41; N,
22
26
2
ꢀ1
1
0
3.25%. IR νmax (KBr, cm ): 3290, 2910,1210. H NMR (CDCl 400 MHz) δ: 7.45ꢀ7.40(m,
3,
4H), 7.08ꢀ7.06(d, J=8.0, 1H), 7.04ꢀ7.02(d, J=8Hz, 1H), 6.78ꢀ6.76 (d, J=8.8Hz, 1H), 6.73ꢀ6.71
(d,J=8.8Hz, 1H), 5.34(s,1H) 4.2(s,1H), 3.73(s, 3H), 3.50(m,1H), 3.01(d,2H), 1.90ꢀ1.18(m,10H).
1
3
C NMR (DMSO, 100MHz): 158.14, 133.04, 132.89, 129.83, 129.66, 128.29, 120.04, 115.97,
113.03, 112.89, 110.87, 86.90, 74.84, 57.02, 56.87, 44.22, 36.46, 25.16, 22.66, 19.23. Mass: m/z
+
found for C H BrNO 416.1, 418.1 ([M+1] ).
2
2
26
2
8

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Synthesis of 4'ꢀ((2ꢀbutylꢀ4ꢀchloroꢀ5ꢀ(5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecanꢀ2ꢀ
yl)ꢀ1Hꢀimidazolꢀ1ꢀyl)methyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀcarbonitrile (5): The compound 5 was
obtained in two steps
Step 1: Preparation of 4'ꢀ((2ꢀbutylꢀ4ꢀchloroꢀ5ꢀformylꢀ1Hꢀimidazolꢀ1ꢀyl)methyl)ꢀ[1,1'ꢀ
2
0
biphenyl]ꢀ2ꢀcarbonitrile: was prepared using reported protocol.
Step 2: Preparation of 4'ꢀ((2ꢀbutylꢀ4ꢀchloroꢀ5ꢀ(5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀ
azaspiro[5.5]undecanꢀ2ꢀyl)ꢀ1Hꢀimidazolꢀ1ꢀyl)methyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀcarbonitrile: This
compound was obtained from AA (1 mmol), 4'ꢀ((2ꢀbutylꢀ4ꢀchloroꢀ5ꢀformylꢀ1Hꢀimidazolꢀ1ꢀ
yl)methyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀcarbonitrile (1 mmol) and K CO (2.5 mmol) in methanol as brown
2
3
0
color crystalline solid, Yield: 88%; Melting point: 59ꢀ60 C; Elemental Analysis Calculated for
C H N ClO : C, 72.95; H, 6.78; N, 09.20; found C, 72.75; H, 6.51; N, 09.14%; IR νmax
37
41
4
2
ꢀ1
1
(
KBr, cm ): 3265, 2914, 1170. H NMR (DMSO 400 MHz) δ: 7.60ꢀ7.47(m,6H), 7.03ꢀ
,
6.97(m,4H), 6.73ꢀ6.70(d, J=8.8,2H), 5.25(s, 1H), 5.10 (s,2H), 4.09 (s, 1H) 3.71(m,1H),
1
3
3
.63(s,3H), 2.9(d,2H), 2.7(t,2H), 1.42ꢀ1.14(m,14H), 0.75ꢀ0.71(t,3H). C NMR (CDCl ,100
3
MHz) δ: 158.24, 151.90, 149.66, 144.99, 137.38, 137.30, 135.09, 133.95, 133.01,132.49,
130.66,130.10, 129.13, 127.81, 126.63, 122.30, 118.73,113.46, 111.35, 86.11, 73.38, 62.61,
56.30, 55.26, 47.96, 36.58, 35.29, 29.61,26.74, 25.84, 22.49, 22.01, 21.87,13.82; Mass: m/z
+
found for C H N ClO : 610.2 ([M+1] ).
3
7
41
4
2
Synthesis
of
5ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀ(2ꢀmethylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ1ꢀoxaꢀ3ꢀ
azaspiro[5.5]undecane (6): Compound 6 was obtained from AA (1 mmol), 2ꢀmethyl indoleꢀ3ꢀ
carbaldehyde (1 mmol) and K CO (2.5 mmol) as colorless crystalline solid, Yield 95 %;
2
3
0
Melting point: 158ꢀ160 C; Elemental Analysis Calculated for C H N O : C, 76.89; H, 7.74;
2
5
30
2
2
ꢀ1
1
N, 07.17; found C, 76.65; H, 7.51; N, 07.14%
.
IR νmax (KBr, cm ): 3270, 3010, 1165, H
9

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NMR (DMSO 400 MHz) δ:11.30(s, 1H), 7.91ꢀ7.89(d, J=8.0Hz,1H), 7.26ꢀ7.25(d, J=7.6Hz,1H),
,
7.18ꢀ7.16(d, J=8.0Hz, 2H),
7.04ꢀ6.98(m,2H),6.78ꢀ6.76(d, J=8.8Hz, 2H), 4.8(s,1H),
4
.2(s,1H),3.79(m,1H), 3.71(s, 3H), 2.90(d, J=2.0Hz, 2H), 2.41(s,3H), 1.64ꢀ1.21(m,10H); 13
C
NMR (CDCl , 100 MHz)δ; 158.27, 140.90, 135.60, 133.93, 130.72, 126.48,122.52, 121.76,
3
120.48, 113.45, 110.71, 110.27, 86.04, 74.63, 63.54, 55.31, 47.01, 38.03, 34.35, 26.08, 22.22,
+
2
2.01, 12.30; Mass: m/z found for C H N O : 391.2 ([M+1] ).
2
5
30
2
2
Synthesis
of
5ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀ(2ꢀphenylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ1ꢀoxaꢀ3ꢀ
azaspiro[5.5]undecane (7) :
Compound 7 was obtained from AA (1 mmol), 2ꢀphenyl indoleꢀ3ꢀcarbaldehyde (1 mmol) and
0
K CO (2.5 mmol) as colorless crystalline solid, Yield: 93%; Melting point: 80ꢀ82 C;
2
3
Elemental Analysis Calculated for C H N O : C, 79.61; H, 7.13; N, 06.19; found C, 79.65; H,
3
0
32
2
2
ꢀ
1
1
7
1
4
.01; N, 06.14% ; IR νmax (KBr, cm ): 3310, 2890,1170, H NMR (DMSO 400 MHz) δ:
,
1.7(s, 1H, ), 8.1(d,2H), 7.6ꢀ7.40(m,2H), 7.4ꢀ7.2(m, 3H), 7.2ꢀ7.0(m, 4H), 6.8(d,2H), 4.5(s,1H),
1
3
.2(s,1H), 3.8(m,1H), 3.7(s,3H), 2.9(d, 2H), 1.7ꢀ1.1(m,10H), C NMR (CDCl , 100MHz)
3
δ:158.24, 133.88, 131.41,130.88, 129.39, 129.09, 129.02, 126.61, 123.73, 122.39, 122.26,
113.79, 113.44, 111.25, 111.05, 86.92, 74.34, 55.32, 50.91, 46.70, 37.97, 26.09, 22.26, 22.03.
+
Mass: m/z found for C H N O :453.3 ([M+1] ).
30
32
2
2
Synthesis of 4'ꢀ((3ꢀ(5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecanꢀ2ꢀyl)indolinꢀ1ꢀ
yl)methyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀcarbonitrile (9): Compound 9 was prepared using the reported
1
9
protocol.
Synthesis
of
4ꢀ(5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecanꢀ2ꢀyl)ꢀN,Nꢀ
dimethylaniline (12): Compound 12 was obtained from AA (1 mmol), 4ꢀ(dimethylamino)
benzaldehyde(1 mmol) and K CO (2.5 mmol) as brown color crystalline solid, Yield: 92%;
2
3
1
0

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0
Melting point: 120ꢀ121 C; Elemental Analysis Calculated for C H N O : C, 75.75; H, 8.48;
2
4
34
2
2
ꢀ1
1
N, 07.36; found C, 75.65; H, 8.51; N, 07.23%
. IR νmax (KBr, cm ): 3280, 2866, 1118, H
NMR (CDCl 400 MHz) δ: 7.51ꢀ7.49(d, J=8.0Hz, 2H), 7.11ꢀ7.09(d, J=8.0Hz, 2H), 6.73ꢀ6.71(d,
3
,
J=8Hz, 2H), 6.60ꢀ6.58(d, J=8.4Hz, 2H), 5.30(s,1H),4.2(s,1H), 3.98ꢀ3.94(m,1H), 3.73(s, 3H),
1
3
2
1
4
.94(s,6H), 2.90ꢀ2.88(d,J=8Hz,2H), 1.77ꢀ1.28(m,10H), C NMR (CDCl ,100 MHz) δ: 158.01,
3
50.41, 135.50, 130.45, 130.10, 128.76, 114.11, 113.90, 112.98, 91.24, 79.21, 56.30, 50.97,
+
7.28, 41.45, 37.10, 26.56, 22.54, 22.34. Mass: m/z found for C H N O : 381.2 ([M+1] ).
24
34
2
2
Synthesis of 3ꢀ(5ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecanꢀ2ꢀyl)ꢀ4Hꢀchromenꢀ4ꢀ
one (13) Compound 13 was obtained from AA (1 mmol), 4ꢀoxoꢀ4Hꢀchromeneꢀ3ꢀcarbaldehyde
1 mmol) and K CO (2.5 mmol) as yellow color crystalline solid, Yield 91%; Melting point:
:
(
2
3
0
5
9ꢀ60 C
; Elemental Analysis Calculated for C H NO : C, 74.05; H, 6.71; N, 03.45; found C,
25 27 4
ꢀ1
1
73.95; H, 6.51; N, 03.34 %
.
IR νmax (KBr, cm ): 3280, 2945,1095, H NMR (DMSO 400
,
MHz) δ:7.66 (s, 1H),7.2ꢀ6.6(m,8H), 5.5(s,1H), 4.1(s,1H),3.75(s,3H), 3.5(m,1H), 2.8(m,2H), 1.7ꢀ
1
3
0
1
4
.9(m,10H); C NMR ( CDCl , 100 MHz) δ: 191.05, 157.52, 157.30, 156.01, 133.50, 132.45,
3
31.12, 130.78, 124.53, 123.42, 118.78, 117.35, 113.15, 112.88, 86.12, 75.21, 56.34, 54.32,
+
7.13, 37.13, 36.40, 25.39, 21.21, 20.30; Mass: m/z found for C H NO 406.2 ([M+1] ).
25
27
4
Synthesis
of
2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀphenoxybenzyl)ꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ5ꢀ(4ꢀ
methoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecane (21): Compound 21 was obtained in two
steps.
Step
1:
Preparation
of
2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀphenoxybenzyl)ꢀ1Hꢀimidazoleꢀ5ꢀ
carbaldehyde: This compound was obtained by using 2ꢀbutylꢀ4ꢀchloroꢀ1Hꢀimidazoleꢀ5ꢀ
carbaldehyde (1 mmol), 1ꢀ(bromomethyl)ꢀ4ꢀphenoxybenzene (1.2 mmol), potassium carbonate
(2.5 mmol), and DMF (8 mL) as a solvent and stirring for 14 h at room temperature.
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1

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Step 2: Preparation of 2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀphenoxybenzyl)ꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ5ꢀ(4ꢀ
methoxyphenyl)ꢀ1ꢀoxaꢀ3ꢀazaspiro[5.5]undecane: This compound was obtained from AA (1
mmol), 2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀphenoxybenzyl)ꢀ1Hꢀimidazoleꢀ5ꢀcarbaldehyde (1 mmol), and
0
K CO (2.5 mmol) in methanol as brown crystalline solid, Yield: 84%; Melting point: 55ꢀ57 C;
2
3
Elemental Analysis Calculated for C H N ClO : C, 72.05; H, 7.05; N, 07.00; found C, 71.95;
36
42
3
3
ꢀ1
1
H, 6.91; N, 06.94%. IR νmax (KBr, cm ): 3290, 2920, 1150; H NMR (CDCl 400 MHz) δ:
3,
7
2
1
5
.4ꢀ6.6(m,13H), 5.4(s, 1H), 5.3(s, 2H), 4.1(s, 1H) 3.75(s, 3H), 3.3(m,1H), 2.60(t,2H),
1
3
.5(d,2H),1.7ꢀ1.2(m,17H); C NMR (100 MHz, DMSOꢀd ) δ: 157.90, 157.10, 154.43, 147.63,
6
40.24, 134.70, 130.78, 129.10, 128.55, 127.93, 122.12, 121.11, 119.23, 115.31, 87.24, 80.46,
6.10, 51.62, 47.65, 36.83, 30.91, 26.43, 25.80, 22.91, 22.40, 14.25; Mass: m/z found for
+
C H N ClO : 600.4 ([M+1] ).
36
42
3
3
Synthesis
of
2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀnitrobenzyl)ꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀ
benzo[d][1,3]oxazine(8): Compound 8 was obtained in two steps.
Step 1: Preparation of 2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀnitrobenzyl)ꢀ1Hꢀimidazoleꢀ5ꢀcarbaldehyde :
This compound was obtained by using 2ꢀbutylꢀ4ꢀchloroꢀ1Hꢀimidazoleꢀ5ꢀcarbaldehyde (1 mmol),
1ꢀ(bromomethyl)ꢀ4ꢀnitrobenzene (1.2 mmol), potassium carbonate(2.5 mmol), and DMF (8 mL)
as a solvent and stirring for 14 h at room temperature.
Step 2: Preparation of 2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀnitrobenzyl)ꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ2,4ꢀdihydroꢀ
1
Hꢀbenzo[d][1,3]oxazine :This compound was obtained from 2ꢀamino benzyl alcohol (1 mmol),
ꢀbutylꢀ4ꢀchloroꢀ1ꢀ(4ꢀnitrobenzyl)ꢀ1Hꢀimidazoleꢀ5ꢀcarbaldehyde (1 mmol) and chloro acetic
2
acid (2.5 mmol) in methanol at room temperature as a brown crystalline solid, Yield 81 %;
0
Melting point 53ꢀ55 C; Elemental Analysis Calculated for C H ClN O : C, 61.90; H, 5.43; N,
2
2
23
4
3
1
1
3.12; found C, 61.84; H, 5.41; N, 13.10%; H NMR (DMSO, 400 MHz) δ: 8.20ꢀ8.18(d,
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2

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J=8.4Hz, 2H), 7.20ꢀ7.13(m, 6H), 5.48(s, 1H), 4.99(s, 2H), 4.60(s,2H), 4.24(s, 1H), 2.67ꢀ2.63(t,
1
3
J=8Hz, 2H), 1.34ꢀ1.24(m, 2H), 0.89ꢀ0.85(t, J=7.2Hz, 3H); C NMR (DMSOꢀd , 100 MHz,) δ:
6
147.81, 144.74, 143.21, 140.75, 135.21, 128.72, 128.01,126.71, 123.10, 122.05, 120.95, 110.96,
9
2.30, 67.31, 46.69, 31.21, 26.31, 22.85, 14.68. Mass: m/z found C H ClN O : 428.14
22
23
4
3
+
(
[M+1] ).
Synthesis of 2ꢀ(2ꢀbutylꢀ4ꢀchloroꢀ1Hꢀimidazolꢀ5ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazine (10),
ꢀchloroꢀ2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazine (11), 3ꢀ(2,4ꢀdihydroꢀ1Hꢀ
benzo[d][1,3]oxazinꢀ2ꢀyl)ꢀ4Hꢀchromenꢀ4ꢀone (14), 2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀ
benzo[d][1,3]oxazine (15), 3ꢀ(6ꢀmethylꢀ2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazinꢀ2ꢀyl)ꢀ4Hꢀchromenꢀ
ꢀone (16), 4'ꢀ((3ꢀ(2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazinꢀ2ꢀyl)ꢀ1Hꢀindolꢀ1ꢀyl)methyl)ꢀ[1,1'ꢀ
biphenyl]ꢀ2ꢀcarbonitrile (17), 6ꢀchloroꢀ2ꢀ(2ꢀphenylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀ
benzo[d][1,3]oxazine (18), 3ꢀ(6ꢀchloroꢀ2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazinꢀ2ꢀyl)ꢀ4Hꢀchromenꢀ
ꢀone (19), 6ꢀmethylꢀ2ꢀ(2ꢀmethylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazine (20), 6ꢀ
methylꢀ2ꢀ(2ꢀphenylꢀ1Hꢀindolꢀ3ꢀyl)ꢀ2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazine (22), 4ꢀ(2,4ꢀdihydroꢀ
7
4
4
1Hꢀbenzo[d][1,3]oxazinꢀ2ꢀyl)phenol (23), and 4ꢀ(7ꢀchloroꢀ2,4ꢀdihydroꢀ1Hꢀbenzo[d][1,3]oxazinꢀ
1
4
2ꢀyl)phenol (24) were prepared using the reported protocol.
Synthesis of 2ꢀ(quinolinꢀ2ꢀyl)phenol (25), 2ꢀ(2ꢀbromophenyl)ꢀ6ꢀmethylquinoline (26), 2ꢀ(2,4ꢀ
dichlorophenyl)Quinoline (27), 4ꢀ(6ꢀchloroquinolinꢀ2ꢀyl)benzenamine (28), and 2ꢀ(2,4ꢀ
1
3
dichlorophenyl)ꢀ6ꢀmethylquinoline (29) were prepared using the reported protocol.
Pharmacology
Human methionylꢀtRNA synthetase inhibition assay. MRS was coꢀexpressed with AIMP3
aminoacylꢀtRNA synthetaseꢀinteracting multifunctional protein 3), which does not affect the
(
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catalytic activity of MRS in vitro, in Escherichia Coli BL21(DE3) to increase protein stability
2
1
and solubility and purified using ProBond Resin (Invitrogen). MRS was eluted in the presence
of 200 mM imidazole (pH 6.0) and dialyzed with PBS containing 20% (vol/vol) glycerol.
Aminoacylation activity of MRS was measured at 37oC in reaction buffer (30 mM HEPES, pH
7.4, 100 mM potassium acetate, 10 mM magnesium acetate, 2mM ATP, 100 µg/mL tRNAi Met,
and 25 µCi [35S] Methionine (1000 Ci/mmol; Izotop)) in the presence of 25 µM or 100 µM
inhibitor. Aminoacylation reactions were quenched on 3 MM filter paper prewetted with 5%
trichloroacetic acid containing 1 mM Methionine. After washing with 5% trichloroacetic acid
and drying, radioactivity was detected by liquid scintillation counter. FmocꢀDLꢀ
selenomethionine was used as positive control for human methionyl tRNA inhibitory studies.
MTT assay. The antiproliferative effect of the compounds synthesized against HCT116 (colon
cancer) and A549 (lung cancer) cells was determined by the MTT dye uptake method as
2
2, 23
4
described previously.
. Briefly, cancer cells (2.5x10 /mL) were incubated in triplicate in a
96ꢀwell plate, in the presence of varying compound concentrations at a volume of 0.2 mL, for
different time intervals at 37 °C. Thereafter, 20 ꢁl MTT solution (5 mg/mL in PBS) was added to
each well. After 2 h incubation at 37 °C, a 0.1 mL lysis buffer (20% SDS, 50%
dimethylformamide) was added; incubation was performed for 1 h at 37 °C, and the optical
density (OD) at 570 nm was measured by plate reader.
Molecular docking studies. Computational docking studies were performed to investigate
molecular interactions between the library of the quinoline, oxazine and benzoxazine ligands and
MRS. Therefore, we prepared the whole set of 34 ligands for docking (addition of explicit
2
4
hydrogens and protonation for pH=7) using MOE and selected the coꢀcrystal structure of MRS
2
5
with methionine and adenosine (PDB: 1PG2) as the target structure. . The protein structure was
1
4

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2
6
prepared using protonate3D whilst the coꢀcrystallized ligand as well as resolved water
molecules were discarded. We selected the position of the C5' atom of adenosine as the cavity
2
7
centre and used GOLD for docking in a radius of 10Å around the centre.
Statistical Analysis. The mean values are expressed ± S.E. for control and experimental samples
and each experiment was repeated a minimum of two times. Statistical significance of data was
determined by applying Student’s t test using the Prism 5 software (GraphPad Software).
Conclusion
Elevated levels of protein synthesis in cancer cells serve as an attractive target for
designing of anticancer agents. Since translation process involves the incorporation of
methionine as first amino acid of all the polypeptides during protein synthesis and inhibition of
Met
esterification of methionine to tRNA by MRS results in collapse of protein synthesis. In order
to provide suitable small molecules to inhibit this process, in the current study we report the
synthesis and biological evaluation of a library of small molecules against human MRS, which
led to the identification of five bioactive molecules. Among the newly synthesized compounds,
compound 21 and compound 8 were presented as the most potent inhibitors of human MRS, and,
on the other hand, both compounds displayed cytotoxicity against cancer cell lines at relatively
subꢀmicromolar concentrations.
Acknowledgements
This research was supported by University Grants Commission (41ꢀ257ꢀ2012ꢀSR), Department
of Science and Technology (NO.SR/FT/LSꢀ142/2012) to Basappa. KSR thanks the Department
of Science and TechnologyꢀPromotion of University Research and Scientific Excellence for
funding. HB & KHK thank UGC for a BSR Fellowship. This work was supported by the Korea
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Health Technology R&D Project through the Korea Health Industry Development Institute
KHIDI) funded by the Ministry of Health & Welfare (HI13C21480301[N.H.K]) and by the
(
Global Frontier program (NRFꢀM3A6A4ꢀ2010ꢀ0029785 [S.K.])
Bibliographic references and notes
1
.
Farhanullah, S. Y. Kim, E. J. Yoon, E. C. Choi, S. Kim, T. Kang, F. Samrin, S. Puri and J. Lee,
Bioorganic & medicinal chemistry, 2006, 14, 7154-7159.
2
3
.
.
A. G. Hinnebusch, Journal of Biological Chemistry, 1997, 272, 21661-21664.
J. P. Kushner, D. Boll, J. Quagliana and S. Dickman, Proceedings of the Society for Experimental
Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.), 1976,
1
53, 273-276.
4
5
.
.
S. Kim, S. You and D. Hwang, Nat Rev Cancer, 2011, 11, 708-718.
S. G. Park, P. Schimmel and S. Kim, Proceedings of the National Academy of Sciences, 2008, 105,
1
1043-11049.
6
7
.
.
M. Bhat, N. Robichaud, L. Hulea, N. Sonenberg, J. Pelletier and I. Topisirovic, Nature reviews.
Drug discovery, 2015, 14, 261-278.
N. H. Kwon, T. Kang, J. Y. Lee, H. H. Kim, H. R. Kim, J. Hong, Y. S. Oh, J. M. Han, M. J. Ku, S. Y. Lee
and S. Kim, Proceedings of the National Academy of Sciences of the United States of America,
2
011, 108, 19635-19640.
T. Hussain, M. Yogavel and A. Sharma, Antimicrobial agents and chemotherapy, 2015, 59, 1856-
867.
8
9
.
.
1
C. F. Crasto, A. K. Forrest, T. Karoli, D. R. March, L. Mensah, P. J. O'Hanlon, M. R. Nairn, M. D.
Oldham, W. Yue, M. G. Banwell and C. J. Easton, Bioorganic & medicinal chemistry, 2003, 11,
2
687-2694.
1
1
1
1
0.
1.
2.
3.
U. A. Ochsner, C. L. Young, K. C. Stone, F. B. Dean, N. Janjic and I. A. Critchley, Antimicrobial
agents and chemotherapy, 2005, 49, 4253-4262.
Q. H. Hu, R. J. Liu, Z. P. Fang, J. Zhang, Y. Y. Ding, M. Tan, M. Wang, W. Pan, H. C. Zhou and E. D.
Wang, Scientific reports, 2013, 3, 2475.
Basappa, C. V. Kavitha and K. S. Rangappa, Bioorganic & medicinal chemistry letters, 2004, 14,
3
279-3281.
H. Bharathkumar, C. D. Mohan, H. Ananda, J. E. Fuchs, F. Li, S. Rangappa, M. Surender, K. C.
Bulusu, K. S. Girish, G. Sethi, A. Bender, Basappa and K. S. Rangappa, Bioorganic & medicinal
chemistry letters, 2015, 25, 1804-1807.
1
1
4.
5.
H. Bharathkumar, M. S. Sundaram, S. Jagadish, S. Paricharak, M. Hemshekhar, D. Mason, K.
Kemparaju, K. S. Girish, Basappa, A. Bender and K. S. Rangappa, PLoS One, 2014, 9, e102759.
Basappa, S. Murugan, C. V. Kavitha, A. Purushothaman, K. G. Nevin, K. Sugahara and K. S.
Rangappa, Cancer letters, 2010, 297, 231-243.
1
1
1
6.
7.
8.
S. Won Lee, Y. Sun Kang and S. Kim, Current Proteomics, 2006, 3, 233-247.
Y.-W. Kim, C. Kwon, J.-L. Liu, S. H. Kim and S. Kim, PloS one, 2012, 7, e40960.
S. Anusha, B. S. Anandakumar, C. D. Mohan, G. P. Nagabhushana, B. S. Priya, K. S. Rangappa,
Basappa and C. G. T, RSC Advances, 2014, 4, 52181-52188.
1
9.
C. D. Mohan, H. Bharathkumar, K. C. Bulusu, V. Pandey, S. Rangappa, J. E. Fuchs, M. K.
Shanmugam, X. Dai, F. Li, A. Deivasigamani, K. M. Hui, A. P. Kumar, P. E. Lobie, A. Bender,
1
6

Page 17 of 23
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB00791G
Basappa, G. Sethi and K. S. Rangappa, The Journal of biological chemistry, 2014, 289, 34296-
4307.
3
2
2
0.
1.
B. Jayashankara and K. Rai, Journal of Chemistry, 2008, 5, 309-315.
T. Kang, N. H. Kwon, J. Y. Lee, M. C. Park, E. Kang, H. H. Kim, T. J. Kang and S. Kim, Journal of
molecular biology, 2012, 423, 475-481.
2
2
2.
3.
H. K. Keerthy, C. D. Mohan, K. Sivaraman Siveen, J. E. Fuchs, S. Rangappa, M. S. Sundaram, F. Li,
K. S. Girish, G. Sethi, Basappa, A. Bender and K. S. Rangappa, The Journal of biological chemistry,
2
014, 289, 31879-31890.
M. Neelgundmath, K. R. Dinesh, C. D. Mohan, F. Li, X. Dai, K. S. Siveen, S. Paricharak, D. J. Mason,
J. E. Fuchs, G. Sethi, A. Bender, K. S. Rangappa, O. Kotresh and Basappa, Bioorganic & medicinal
chemistry letters, 2015, 25, 893-897.
2
2
4.
5.
C. C. G. I. Molecular Operating Environment (MOE) and Q. Montreal, Canada, 2013.
T. Crepin, E. Schmitt, Y. Mechulam, P. B. Sampson, M. D. Vaughan, J. F. Honek and S. Blanquet,
Journal of molecular biology, 2003, 332, 59-72.
2
2
6.
7.
P. Labute, Proteins, 2009, 75, 187-205.
G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, Journal of molecular biology, 1997, 267,
7
27-748.
Schemes
1
7

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Scheme 1: schematic representation for the synthesis of 1,3ꢀoxazines: (i)cyclohexanone,
NaOH, Bu NBr, water–MeOH, RT, 15 h, 96%; (ii) RaneyꢀNi, H (10 atm), anhydrous NH ,
4
2
3
0
MeOH, 35–40 C, 3 h. (iii) RꢀCHO , anhydrous K CO /CH OH, RT, 9 h, (Yield: 85ꢀ95%)
2
3
3
Scheme 2: Schematic representation for the synthesis of benzoxazines (Yield: 85ꢀ96%).
Scheme 3: Schematic representation for the synthesis of quinolines (Yield: 86%).
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8

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Figure Legends
Figure 1: Screening of new compounds (1ꢀ34) against the activity of human methionylꢀtRNA
synthetase at 100 µM. Data are represented as mean ± S.E. (n = 3). PC, positive control, Fmocꢀ
DLꢀselenomethionine; *, P < 0.05; **, P < 0.01.
Figure 2: The lead molecules, which exhibited inhibitory activity against human methionylꢀ
tRNA synthetase at 100 µM were chosen and evaluated at 25 µM and 100 µM.
Figure3: Predicted molecular interactions of the most active compound 21 and methionylꢀtRNA
synthetase (from PDB 1PG2). Left: Native contacts of the ligands (elemental colours with carbon
in grey) and MRS (green cartoon). Sideꢀchains of key amino acids (Asp296, Trp253, Tyr15,
His24) are highlighted as lines. Right: predicted binding mode for compound 21, that shows
major volume overlap with the native ligands. The central oxazine moiety is interacting with
Aspꢀ296, whereas substituents form multiple hydrophobic contacts and πꢀπ interactions.
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9

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Table 1: Cytotoxic studies of lead compounds (against HCT116 and A549 cells) that targets
human MRS.
HCT116
IC50
(µM)
A549
Lead
Compounds
IC (µM)
5
0
Compound 8 19.8 ± 4.9 41.9 ± 5.7
Compound 19 >50 >50
Compound 21 17.7 ± 3.9 28.4 ± 6.1
Compound 23 49.1 ± 7.8
Compound 26 41.3 ± 4.6
Compound 29 40.4 ± 5.5
>50
>50
>50
Paclitaxel
0.0054
0.0046
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0

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Figure 1: Screening of new compounds (1ꢀ34) against the activity of human methionylꢀtRNA synthetase at
1
00 µM. Data are represented as mean ± S.E. (n = 3). PC, positive control, FmocꢀDLꢀselenomethionine; *,
P < 0.05; **, P < 0.01.
2
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Organic & Biomolecular Chemistry
Page 22 of 23
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DOI: 10.1039/C5OB00791G
The lead molecules, which exhibited inhibitory activity against human methionylꢀtRNA synthetase at 100 µM
were chosen and evaluated at 25 µM and 100 µM.
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Page 23 of 23
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C5OB00791G
3
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