Design, synthesis and docking studies of novel 1,2-dihydro-4-hydroxy-2-oxoquinoline-3-carboxamide derivatives as a potential anti-proliferative agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.062

A new series of 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamide hybrids 8a-l have been designed and synthesized using peptide coupling agents with substituted N-phenyl piperazines and piperidines with good to excellent yields. The synthesized compounds were evaluated for their in?vitro anti-proliferative activity against PANC 1, HeLa and MDA-MB-231. The compounds 8d, 8e, 8f, 8g, 8h and 8k exhibited considerable anti-proliferative activity with GI₅₀values ranging from 0.15 to 1.4?μM. The structure and anti-proliferative activity relationship was further supported by in silico molecular docking study of the active compounds against tubulin protein.

Full text of DOI:10.1016/j.ejmech.2016.09.062

Accepted Manuscript
Design, synthesis and docking studies of novel 1,2-dihydro-4-hydroxy-2-
oxoquinoline-3-carboxamide derivatives as a potential anti-proliferative agents
Saleha Banu, Rajitha Bollu, Rajashaker Bantu, Lingaiah Nagarapu, Sowjanya
Polepalli, Nishant Jain, Radhika Vangala, Vijjulatha Manga
PII:
S0223-5234(16)30799-1
10.1016/j.ejmech.2016.09.062
EJMECH 8928
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 April 2016
Revised Date: 19 September 2016
Accepted Date: 20 September 2016
Please cite this article as: S. Banu, R. Bollu, R. Bantu, L. Nagarapu, S. Polepalli, N. Jain, R. Vangala,
V. Manga, Design, synthesis and docking studies of novel 1,2-dihydro-4-hydroxy-2-oxoquinoline-3-
carboxamide derivatives as a potential anti-proliferative agents, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.062.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, synthesis and docking studies of novel
,2-dihydro-4-hydroxy-2-oxoquinoline-3-
Leave this area blank for abstract info.
1
carboxamide derivatives as potential anti cancer
agents
a
a
a
a
b
b
Saleha Banu , Rajitha Bollu , Rajashaker Bantu , Lingaiah Nagarapu *, Sowjanya Polepalli , Nishant Jain ,
c
c
Radhika Vangala , Vijjulatha Manga .
A new series of 1,2-dihydro-4-hydroxy-2-oxoquinoline-3-carboxamide derivatives have been synthesized and
evaluated for their anti-proliferative activity against three human cancer cell lines of PANC 1, HeLa and MDA-
MB-231; showed moderate to good activity.

ACCEPTED MANUSCRIPT
Design, synthesis and docking studies of novel 1,2-dihydro-4-
hydroxy-2-oxoquinoline-3-carboxamide derivatives as a potential
anti-proliferative agents
a
a
a
a
b
Saleha Banu , Rajitha Bollu , Rajashaker Bantu ,Lingaiah Nagarapu *, Sowjanya Polepalli ,
b
c
c
Nishant Jain ,Radhika Vangala , Vijjulatha Manga .
a
Organic Chemistry Division II (CPC), CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500007, India.
Center for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500007, India.
b
c
Molecular Modeling and Medicinal Chemistry Group, Department of Chemistry, Osmania University, Tarnaka,
Hyderabad-500007, India.
ABSTRACT
A new series of 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamide
hybrids 8a-l have been designed and synthesized using peptide coupling agents with
substituted N-phenyl piperazines and piperidines with good to excellent yields. The
synthesized compounds were evaluated for their in vitro anti-proliferative activity against
PANC 1, HeLa and MDA-MB-231. The compounds 8d, 8e, 8f, 8g, 8h and 8k exhibited
considerable anti-proliferative activity with GI50 values ranging from 0.15 to 1.4 µM. The
structure and anti-proliferative activity relationship was further supported by in silico
molecular docking study of the active compounds against tubulin protein.
Keywords:
Roquinimex,
1,2-dihydro-4-hydroxy-2-oxoquinoline-3-carboxamides,
piperazines, anti-proliferative, multiple sclerosis, cell lines and molecular modeling.
1
. Introduction
Quinolines and their hetero fused scaffolds are prevalent molecular structures in
chemistry, biology and medicine [1-3]. Among the various types of substituted quinolines,
,2-dihydro-4-hydroxy-2-oxoquinoline-3-carboxamide derivatives gained specific
importance as privileged medicinal scaffolds (Fig.1). For example, Roquinimex (Linomide)
A), a novel immuno modulator, has been shown to be effective against various types of
1
(
cancers [4, 5]and autoimmune diseases such as MRL/l mice [6], (NZB_NZW) F1 hybrid
mice [7] and experimental autoimmune encephalomyelitis [8, 9]. Recent clinical trials
suggest that (A) has potential in the treatment of autoimmune disease such as rheumatoid
arthritis, systemic lupus erythematosis and multiple sclerosis [10-12]. Interestingly,
Tasquinimod (B) is a lead second generation quinoline-3-carboxamide linomide analogue
1

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which is anti-angiogenic agent for the treatment of prostate cancer , which produces robust
and consistent in vivo growth inhibition as well as suppression of metastasis in a large series
of pre-clinical human xenograft and rodent prostate cancer models [13,14]. Laquinimod (C)
is an oral drug in clinical trials for the treatment of multiple sclerosis (MS) which was
developed by Active Biotech Research AB with linomide as a lead compound [15,16] and
has successfully undergone a clinical phase II trial as multiple sclerosis medication[17].
Owing to the importance of these scaffolds, synthesis and functionalization of 1,2-dihydro-4-
hydroxy-2-oxoquinoline-3-carboxamide compounds attract great interest.
<<< Fig-1>>>
Literature survey informs that piperazines and substituted piperazines are important
family of heterocyclic compounds as they have attracted significant interest in medicinal
chemistry [18-20] and also some piperidine derivatives are important due to their
antimicrobial properties [21]. Many currently notable drugs contain a piperazine ring as a part
of their molecular structure as shown in Fig.2.
<<<Fig-2>>>
The molecular manipulation of promising lead compounds is still a major line of
approach to develop new drugs. It involves an effort to combine the separate pharmacophoric
groups of similar activity into one compound, thus making structural changes in the
biological activity. Considering the above facts, it was thought worthwhile to synthesize the
compounds with a view to obtain certain chemical entities with active pharmacophores in a
single molecular frame work [22-24].
As a part of our endeavor towards new and efficient anticancer agents [25], we herein
report an efficient method for the synthesis of novel 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-
(piperdine/ piperazin-1-yl)propyl)-1,2-dihydroquinoline-3-carboxamide hybrids 8a-l in good
to excellent yields. The synthesized hybrids 8a-l were evaluated for their in vitro anti-
proliferative activity against three human cancer cell lines such as PANC 1 (pancreatic),
HeLa (cervical) and MDA-MB-231 (breast) using a SRB cell proliferation assay to estimate
the viability or growth. Significantly, the compounds 8d, 8e and 8k showed promising anti-
proliferative activity with GI values ranging from 0.15 to 1.4 µM, 0.30 to 0.94 µM and 0.51
5
0
to 1.3µM respectively against all three human cancer cell lines. The structure and anti-
proliferative activity relationship was further supported by in silico molecular docking study
of the active compounds against tubulin protein. Numerous molecules were reported as
tubulin polymerization inhibitors where some of them were quinoline derivatives [26-28].
2

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2
. Results and discussion
2
.1 Chemistry
The
synthesis
of
the
desired
4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-
(piperdine/piperazin-1-yl)propyl)-1,2-dihydroquinoline-3-carboxamideswas performed in six
steps starting from isatoic anhydride using diethyl malonate and β-alanine ester.
The first synthetic step involved the subsequent N-methylation of isatoic anhydride
with iodomethane in presence of sodium hydride in DMF about 5h at room temperature to
give the corresponding N-methylated isatoic anhydride (2). The corresponding N-methylated
isatoic anhydride was then condensed with diethyl malonate in presence of sodium hydride at
o
1
20 C in DMF solution for 12 h and refluxed to get the compound, ethyl 4-hydroxy-1-
methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (3), which was further hydrolysed in
o
presence of glacial acetic acid and hydrochloric acid at 85 C for 4 h to get the targeted
compound, 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (4), in
quantitative yield (Scheme 1).
<<< Scheme 1>>>
Treatment of 1,2-dihydroquinoline-3-carboxylic acid with β-alanine ester in the
presence of HOBt, EDC.HCl and triethylamine at room temperature for 16 h gave ethyl 3-(4-
hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)propanoate (5), which was
further hydrolysed using KOH, ethanol at reflux temperature for 4 h to get 3-(4-hydroxy-1-
methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)propanoic acid (6). Treatment of
compound 6 with substituted piperazines and secondary amines (7a-l) in the presence of
HOBT, EDC.HCl and triethyl amine at room temperature for 24 h gave the targeted 4-
hydroxy-1-methyl-2-oxo-N-(3-oxo-3-(piperdine/piperazin-1-yl)propyl)-1,2-dihydroquinoline-
3
-carboxamides 8a-l in good yields (Scheme 2) (Table 1).
<< Scheme 2>>>
<<Table 1>>>
<
<
The synthesized 1,2-dihydroquinoline-3-carboxamides 8a-l were subjected to
1
analytical characterization and confirmed on the basis of their spectral data. In H NMR
spectra, the characteristic triplet signals appeared for piperazine protons at δ 3.10-3.85 ppm.
The structures for all these compounds were further confirmed by HRMS analysis. For
+
instance, 8a displayed a molecular ion peak at m/z453.19246[M+H] suggesting the
molecular formula of C H O N F.Additionally, the IR spectra for the target compounds
2
4
25
4
4
3

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-1
-1
8
a-l exhibited characteristic absorption bands at 1629-1633 cm ,3060-3300 cm and 2853-
-1
2
952 cm which corresponded to C=O, N-H and C-H respectively.
3
2
.2Pharmacological evaluation.
2
.2.1Effects of the compounds on the viability of human cancer cells.
The in vitro anti-proliferative activity of the designed compounds 8a-l were evaluated
against a panel of three different human cancer cell lines, PANC 1 (pancreatic), HeLa
cervical) and MDA-MB-231 (breast) summarized in Table 2. The compounds were picked
(
for an advanced assay against these three human cancer cell lines at five different
concentrations (0.01, 0.1, 1, 10, 100 µM). GI (growth inhibitory activity) was calculated
5
0
and these values corresponded to the concentration of the compound causing 50% decrease in
the net cell growth as compared to the standard drugs, Nocodazole and Combretastatin.
Results were calculated for each of these parameters if the level of activity was reached;
however, if the effect was not achieved, the value was expressed as greater or less than the
maximum or minimum concentration tested.
Based on Table 2, the synthesized compounds 8a-l showed significant to moderate
cancer cell growth inhibition with GI50 values ranging from 0.15 to 5.4µM. The effect of
various substituent's attached to 1,2-dihydroquinoline-3-carboxamido propanoic acid (6) was
examined. In particular, the compounds 8d, 8e, 8f, 8g, 8h and 8k showed promising anti-
proliferative activity with GI50 values ranging from 0.15-1.40µM respectively, against the
three human cancer cell lines. Among them, compounds 8d, 8e and 8k showed good anti-
proliferative activity against all the three human cancer cell lines.
The biological assessment results against PANC 1 (pancreatic) cell line indicated that
the piperdine analogue 8d exhibited good anti-proliferative activity against this particular cell
line. When the activities of N-alkyl piperazines and N-phenyl piperazines were compared, the
th
analogue 8e with nitro group incorporated into the 4 position of phenyl ring of piperazine
moiety attached to (6) contributed promising anti-proliferative activity against all three cell
lines. The compound 8e showed significant anti-proliferative activity against MDA-MB231.
According to the data of biological research regarding the potency of the phenyl substituted
compounds, the presence of nitro and methoxy groups to the phenyl ring of piperazine moiety
with (6) displayed promising anti-proliferative activity against all the three cancer cell lines.
Moreover we noticed that the compounds 8h and 8k displayed similar results against
all the three cell lines indicating there was no significant change in their activities due to the
4

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changes in position of the methoxy functional groups in the phenyl ring of piperazine moiety.
The halogenated piperazine bearing analogues also showed considerable anti-proliferative
activity against all the human cancer cell lines. The results showed that the substitutions on
the ring attached to 1,2-dihdroquinoline-3-carboxamido propanoic acid have influence on the
potency of anti-proliferative activity against all the three cancer cell lines. Fig.3.
<<<Fig-3>>>
<<<Table2>>>
2
.2.2 Molecular Docking studies:
Microtubules are the key components of the cytoskeleton of eukaryotic cells and has an
important role in various cellular functions such as intracellular migration and transport, cell
shape maintenance, polarity, cell signalling and mitosis. It plays a critical role in cell division
by involving in the movement and attachment of the chromosomes during various stages of
mitosis. Therefore, microtubule dynamics is an important target for the developing anti-
cancer drugs.
Combretastatin is a natural phenol that specifically binds to cochicine binding site of
tubulin for its anti-mitotic effects. Based on the literature evidence the quinolines and
dihydroquinolines have been reported as tubulin inhibitors. Hence in the present study we
have docked the current series of compounds along with the combretastatin in the tubulin
protein to understand the probable binding mode and key active site interactions.
From the docking studies we observed that combretastatin showed hydrogen bond
interactions with Val 238 and Ala 317 and hydrophobic interactions with Ala 250, Leu 255,
Ala 316 and Lys 352 residues. The compounds 8d showed hydrophobic interactions with Val
2
38, Leu 242, Leu 252, Lys 254, Leu 255 and Lys 352 and 8e showed hydrophobic
interactions with Val 238, Leu 242, Leu 255, Ala 316, Lys 352 and Ileu 378 residues. Figure
represents the docked poses of combretastatin, 8d and 8e molecules in the tubulin active
site. The GI and pGI values, and dock scores were given in Table 3.
4
5
0
50
<<< Fig-4>>>
<<< Table 3>>>
From the above results we have observed that the most potent compounds 8d and 8e
showed some common interactions ( Val 238, Leu 255, Lys 352) as compared to
combretastatin which is in agreement with experimental anti-proliferative activities. The
5

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other compounds also showed comparable binding and good dock scores. To validate the
correlation between anti-proliferative activity and the dock score a regression analysis was
performed. The correlation between experimental activity and dock scores is greater than
5
0% [29]. Dock score (glide score) and GI of PANC1, HeLa and MDA-MB-231 gave a
50
correlation coefficient values (r) of -0.58, -0.52 and -0.52 which shows significant relation
between biological activity and docking. The scatter plots of pGI of PANC1, HeLa, MDA-
5
0
MB-231 and their dock scores are shown in Fig.5.
<<< Fig-5>>>
Experimental molecular 3D structures of ligands checked for their ADME properties
using QikProp4.0. (Table 4). ADME describes about Absorption, Distribution, Metabolism
and Excretion of drug candidates. These are considered as drug like properties need to fulfil
by the drug molecule to pass through phase-I clinical trials.
They are defined as
Absorption (A): Process of entering the drug substance into the blood stream
in silico parameters supported are logS and logP
Distribution (D): Dispersion of drug to other parts of the body defined by logBB
Metabolism (M): Biotransformation, irreversible transformation of parent compounds into
active metabolites
Excretion (E): Removal of drug metabolites from the body.
QikProp helps in analyzing pharmacokinetics and pharmacodynamics of the ligand by
accessing drug like properties. It predicts both physically significant descriptors and
pharmaceutically relevant properties. Neutralized analogues of molecules are used for
calculating ADME properties. Properties consists of principal descriptors and the
physiochemical properties with a detailed analysis of the predicted octanol/water partition
coefficient (QPlogPo/w) and water solubility (QPlogS), critical for estimating the absorption
and distribution of drugs within the body. % human oral absorption in intestine, predicted
brain/blood partition coefficient (QPlogBB), predicted apparent Caco-2 cell permeability in
nm/sec (QPPCaco) and predicted apparent MDCK cell permeability in nm/sec (QPPMDCK).
Caco-2 cells are a model for the gut-blood barrier whereas MDCK cells are considered to be
a good mimic for the blood-brain barrier. It also evaluates an acceptability of the analogues
based on the Lipinski's rule of 5 (number of violations of Lipinski’s rule of five) that is
essential for rational drug design. Poor absorption or permeation are more likely when a
6

ACCEPTED MANUSCRIPT
ligand molecule violates Lipinski’s rule of five i.e., more than 5 hydrogen bond donors, the
molecular weight over 750, the log P over 5 and the sum of N’s and O’s over 10.
The calculated values for the synthesized molecules ranged within the acceptable
limits.
<<< Table4>>>
Crossing the blood-brain barrier (BBB), which is a prerequisite for the entry of drugs
to CNS, was found to be in the acceptable range indicating that the compounds may be
considered for further development. The predicted percentage human oral absorption for all
the molecules also in acceptable range.
3
. Conclusion
Taken together, we synthesized a series of novel 4-hydroxy-1-methyl-2-oxo-N-(3-
oxo-3-(piperazin-1-yl) propyl)-1,2-dihydroquinoline-3-carboxamides 8a-l in good yields and
performed anti-proliferative activity against three different human cancer cell lines, namely
PANC 1, HeLa, and MDA-MB-231 (pancreatic, cervical and breast respectively). Among the
tested cancer cell lines, Compounds 8d, 8e, 8f, 8g, 8h, and 8k exhibited promising anti-
proliferative activity with GI values ranging from 0.15 to 1.4 µM against all cell lines, like
5
0
PANC 1, HeLa and MDA-MB-231. Significantly, the compounds 8d showed significant
activity against PANC 1with GI50 value 0.15 µM, 8e showed significant activity against
MDA-MB-231 with GI50 value 0.30 µM and 8k showed significant activity against HeLa
with GI50 value 0.73 µM. Based on the biological assay, molecular docking and ADME
studies, the synthesized molecules can be considered as promising lead molecules for the
development of new anti cancer agents.
4
4
. Experimental protocols
.1. Biological Study
Materials and Methods.Cell Cultures, Maintenance and Anti proliferative Evaluation
The cell lines, PANC 1, HeLa, and MDA MB 231 (pancreatic, cervical and breast) used in
this study was procured from American Type Culture Collection (ATCC), USA. The
synthesized test compounds were evaluated for their in vitro anti-proliferative activity in
these three different human cancer cell lines. A protocol of 48 h continuous drug exposure
was used and an SRB cell proliferation assay was used to estimate cell viability or growth.
7

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All the cell lines were grown in Dulbecco's modified Eagle's medium (containing 10% FBS
in a humidified atmosphere of 5% CO at 37°C). Cells were trypsinized when sub-confluent
2
from T25 flasks/60 mm dishes and seeded in 96-well plates in 100 ꢀL aliquots at plating
densities depending on the doubling time of individual cell lines. The microtitre plates were
incubated at 37 °C, 5% CO , 95% air, and 100% relative humidity for 24 h prior to the
2
addition of experimental drugs and were incubated for 48 h with different doses (0.01, 0.1, 1,
1
0, 100 µM) of the prepared derivatives.After incubation at 37 °C for 48 h, the cell
monolayers were fixed by the addition of 10% (wt/vol) cold trichloroacetic acid and
incubated at 4 °C for 1 h and were then stained with 0.057% SRB dissolved in 1% acetic acid
for 30 min at room temperature. Unbound SRB was washed with 1% acetic acid. The
protein–bound dye was dissolved in 10 mM Tris base solution for OD determination at 510
nm using a microplate reader (Enspire, Perkin Elmer, USA). Using the seven absorbance
measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at
the five concentration levels (Ti)], the percentage growth was calculated at each of the drug
concentrations levels. Percentage growth inhibition was calculated as:
[
[
(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz
(Ti-Tz)/Tz] x100 for concentrations for which Ti<Tz.
The dose response parameter, growth inhibition of 50% (GI ) was calculated from [(Ti-
5
0
Tz)/(C-Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the
net protein increase (as measured by SRB staining) in control cells during the drug
incubation. Values were calculated for this parameter if the level of activity is reached;
however, if the effect is not reached or is exceeded, the value for that parameter was
expressed as greater or less than the maximum or minimum concentration tested.
4
.2 Molecular modeling
Docking studies: Molecular docking studies were carried out using Glide version 5.6.
30] Crystal structure of β-tubulin (PDB ID: 1SA0) [31] was downloaded from protein data
[
bank. 12 molecules were docked into 1SA0. The hydrogen atoms were added and unwanted
water molecules were removed from the protein structure. The receptor binding site was
defined as a Glide enclosing box in the centroid of the co-crystallized ligand molecule and
the size was set to a default value of 26Å without any hydrogen bonding constraint.
Ligand preparation: LigPrep [32] was used to attach hydrogen, converts 2D structures
to 3D, generates stereoisomer and optionally neutralizes charged structures or determines the
8

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most probable ionization state at user-defined pH. All the structures were ionized at neutral
pH 7. Conformers for each ligand were generated using ConfGen by applying OPLS-2005
force field method. [33]
4
.3 General information
All the solvents and reagents were purchased from commercial suppliers and were used
without further purification. Melting points were measured with a Fischer-Johns melting
point apparatus and were uncorrected. Nuclear Magnetic Resonance spectra were recorded on
3
00 (Bruker) and 500 MHz (Varian) spectrometers in appropriate solvents using TMS as
internal standard or the solvent signals as secondary standards and the chemical shifts are
represented in scales. Multiplicities of NMR signals are designated as s (singlet), d
doublet), doublet of doublet (dd), t (triplet), m (multiplet, for unresolved lines), etc.
δ
1
3
(
C
NMR spectra were recorded on 75 and 125 MHz spectrometer. IR spectra were recorded on
Perkin-Elmer model 683 or 1310 spectrometers with sodium chloride optics or KBr pellets
with neat. ESI-MS were recorded on Thermo Finnigan LCQ ion trap mass spectrometer
equipped with electron spray ionization. High-resolution mass spectra were obtained by using
ESI-QTOF mass spectrometer. All the experiments were monitored by analytical thin layer
chromatography (TLC) performed on silica gel GF254 pre-coated plates. After elution, the
plate was visualized for the spots on TLC plates which was achieved either by exposure to
UV (254 nm) light, iodine vapour and/or by dipping the plates in phosphomolybdic acid-ceric
(
IV) sulfate-sulfuric acid solution (PMA solution) and heating the plates at 110ºC.Solvents
were removed under vacuum and heated in a water bath at 40°C. Silica gel (60-120 mesh)
was used for column chromatography. Columns were packed as the slurry of silica gel in
hexane and equilibrated with the appropriate solvent/solvent mixture prior to use. The
compounds were loaded neat or as a concentrated solution using the appropriate solvent
system. Appropriate names (if possible) for all the new compounds were given with the help
of ChemBioOffice v12.0; 2012.
4
.4 Synthesis of 1-methyl-1H-benzo[d][1, 3] oxazine-2, 4-dione (2):
Isatoic anhydride (12.26 mmol) in DMF (30 mL) was taken, sodium hydride (18.3
mmol) was added portion wise and stirred for 10 min. Reaction mixture was cooled to 0 °C,
lodomethane (18.3 mmol) was added drop wise and stirred at RT for 5 h. Reaction was
monitored by TLC. After completion of reaction, ice water was added, extracted with ethyl
acetate. Organic layer was dried over anhydrous sodium sulfate and evaporated to dryness.
9

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Crude product was purified by column chromatography using an eluent of 35% ethyl acetate
in hexane to afford 1 -methyl- l H-benzo[d][ l ,3]oxazine-2,4-dione as brown coloured solid
1
with 64% yield. HNMR (400 MHz, DMSO d ): δ 3.59 (s, 3H, CH ), 7.30-7.39 (m, 1 H, Ar-
6
3
H), 7.79-7.86 (m, 1 H, Ar-H), 8.08-8.13( m, 1H, Ar-H).
4
.5 Synthesis of ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (3):
A solution of diethyl malonate (51 mmol) was added slowly to a suspension of
sodium hydride (60percent in mineral oil, 56 mmol) in dimethylformamide under N2
atmosphere. The mixture was stirred at room temperature until the evolution of hydrogen gas
o
ceased, then heated to 90 C for 30 minutes and cooled to room temperature. A solution of N-
methylisatoic anhydride (56 mmol) in dimethylformamide was added slowly and the mixture
o
was heated overnight at 120 C. The mixture was then cooled to room temperature, poured
into ice water, and acidified by cold 10% HCI. The solids formed were filtered and washed
several times by water to afford ethyl 4-hydroxy-1-methyl-2-oxo-1, 2-dihydroquinoline-3-
1
carboxylate as light brown coloured solid with 78% yield. H NMR (500 MHz, CDCl ): δ1.49
3
(
t,J = 7.1 Hz, 3H, CH ),3.66 (s, 3H, CH ), 4.51 (q, J = 7.1 Hz, 2H, CH ), 7.27-7.38 (m, 1H,
3 3 2
Ar-H), 7.66-7.73 (m, 1H, Ar-H), 8.19 (dd, J = 7.9 Hz, 1.5 Hz, 1H, Ar-H), 14.20 (s,1H, OH).
4
.6 Synthesis of 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (4):
1
,2-Dihydro-4-hydroxy-l-methyl-2-oxo-3-quinoline carboxylic acid methyl ester
0
(
3.61 mmol) was added to glacial acetic acid (88.6 mmol) at 25-30 C followed by the
0
addition of hydrochloric acid (54.5mmol) and then heated to 80-85 C. The reaction mixture
0
was stirred at 80-85 C for 6 hours. The resulting mass was cooled to 10-15 °C followed by
the addition of ethanol (10 mL). The resulting solid was filtered and washed with ethanol (10
ml) and then dried to get l,2-dihydro-4-hydroxy-l-methyl-2-oxo-3- quinoline carboxylic acid
1
as white coloured solid with 86% yield. H NMR (500 MHz, CDCl ): δ3.77 (s, 3H, CH ),
3
3
7
.38-7.54 (m, 2H, Ar-H), 7.77-7.87 (m, 1H,Ar-H), 8.27 (dd, , J = 8.0 Hz, 1.2 Hz, 1H, Ar-H),
1
4.55 (s, 1H, COOH), 15.60 (s,1H,OH).
4
.7
Synthesis
of
ethyl
3-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-
carboxamido) propanoate (5):
1
0

ACCEPTED MANUSCRIPT
Compound 4 (1mmol) was dissolved in freshly distilled dichloromethane (5 mL)
under nitrogen atmosphere. Then HOBt (1.1 mmol), EDC.HCl (1.1mmol) were added to it.
The reaction mixture was stirred for 5 minutes, and β-alanine ethyl ester hydrochloride (1.5
mmol) was added slowly, followed by the addition of triethylamine (2 mmol) and stirred for
1
6 h at room temperature. The progress of the reaction was monitored by TLC. After
completion of the reaction, water (10 mL) was added to the reaction mixture and extracted
with dichloromethane. Organic layer was collected and dried over anhydrous Na SO
2
4.
Solvent was removed under reduced pressure and the resultant compound was recrystallized
1
with ether to afford the title compound 5 as pale brown solid with 76% yield. H
NMR(500MHz, CDCl ): 1.29 (t, J=7.1Hz, 3H, CH ) 2.67 (t, J =6.5Hz, 2H, CH ), 3.68 (s, 3H,
3
3
2
CH ), 3.74 (q, J= 6.4 Hz, 2H, CH ), 4.20 (q, J = 7.1 Hz, 2H, CH ), 7.28-7.33 (m, 1H, Ar-H),
3
2
2
7
.34-7.38 (m, 1H, Ar-H), 7.65-7.72 (m, 1H, Ar-H), 8.21 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 10.59
(t, J =6.7Hz, 1H, CONH).
4
.8 Synthesis of 3-(4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxamido)
propanoic acid (6):
The compound 5 (1.2 mmol) was dissolved in ethanol (10 mL), added 1N potassium
hydroxide solution (10 mL) and the reaction mixture was refluxed for 4 h, and completion of
the reaction was monitored by TLC. After completion of the reaction, the reaction mixture
was allowed to cool to room temperature, neutralized with diluted hydrochloric acid and the
solid separated was filtered through Buckner funnel and washed with water and dried over
reduced pressure to obtain the crude compound. The above crude product was recrystallized
1
from ethanol to obtain the title compound 6 as white solid with 74% yield.
H
NMR(300MHz, CDCl ): 2.75 (t, J= 6.4 Hz, 2H, CH ), 3.69 (s, 3H, CH ), 3.72-3.80 (m, 2H,
3
2
3
CH ), 7.30-7.38 (m, 2H, Ar-H), 7.65-7.73(m, 1H, Ar-H), 8.23 (dd, J = 7.7 Hz,1.37 Hz, 1H,
2
1
3
Ar-H), 10.62 (t, J = 5.6Hz, 1H, CONH). C NMR (75 MHz, CDCl ): δ20.07, 32.69, 34.95,
3
9
6.71, 114.09, 122.17, 125.36, 133.55, 162.50, 139.83, 162.50, 170.99, 179.50; MS (ESI):
+
m/z 291[M+H] ;
4
.9General procedure for the synthesis of compounds8a-l:
Compound 6 (1mmol) was dissolved in freshly distilled dichloromethane (5mL) under
nitrogen atmosphere. Then HOBt (1.1 mmol), EDC,HCl (1.1mmol) were added to it. The
reaction mixture was stirred for 5 minutes, secondary amine 7a-l (1.2 mmol) was added in a
1
1

ACCEPTED MANUSCRIPT
slow stream and followed by the addition of triethylamine (2 mmol) and the reaction mixture
was stirred for 24 h at room temperature. The progress of the reaction was monitored by
TLC. After completion of the reaction, water (10 mL) was added to the reaction mixture and
extracted with dichloromethane. Organic layer was collected and dried over anhydrous
Na SO After filtration, the solvent was removed under reduced pressure and the crude
2
4.
product was purified by silica gel chromatography using an eluent of 50% ethyl acetate in
hexane.
4
1
.9.1 N-(3-(4-(2-fluorophenyl)piperazin-1-yl)-3-oxopropyl)-4-hydroxy-1-methyl-2-oxo-
,2-dihydroquinoline-3-carboxamide (8a):
o
Light brown solid (82% yield): m.p: 160-164 C; IR (KBr, ν ): 3090, 2924, 2854, 1633,
-
1 1
1
2
3
574, 1504, 1442, 1231, 1021, 803,764cm ; H NMR (500 MHz, CDCl ): δ2.73 (t, J=6.4 Hz,
3
H, CH ), 3.04-3.09 (m, 4H, 2CH ), 3.64 (t, J=5.2 Hz, 2H, CH ), 3.68 (s, 3H, CH ), 3.78-
2
2
2
3
.82 (m, 2H, CH ), 3.82-3.85 (m, 2H,CH ), 6.89-6.99 (m, 2H, Ar-H), 7.01-7.07 (m, 2H, Ar-
2
2
H), 7.28-7.33 (m, 1H, Ar-H), 7.34-7.37 (m, 1H, Ar-H), 7.67-7.71 (m, 1H, Ar-H), 8.22 (dd, J=
1
3
8
.0Hz, 1.3Hz, 1H, Ar-H), 10.64 (t, J= 5.5 Hz, 1H, CONH); C NMR (75 MHz, CDCl3):
δ29.65, 32.77, 45.50, 50.33, 50.78, 96.83, 114.16, 116.07, 119.14, 122.25, 123.06, 124.46,
+
1
33.66, 139.93, 162.51, 169.38, 171.11, 171.81; MS (ESI):m/z 453 [M+H] ; HRMS: Calcd
for C H O N F: 453.19326, Found : 453.19246.
2
4
25
4
4
4
.9.2 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-(4-phenylpiperazin-1-yl)propyl)-1,2-
dihydroquinoline-3-carboxamide (8b):
Light brown solid (84% yield) : m.p:162-166 C; IR (KBr, ν): 3379, 2925, 2858, 1634,
o
-
1 1
1
2
3
1
494, 1441, 1230, 1020, 803, 764 cm ; H NMR (500 MHz, CDCl ): δ 2.73 (t, J= 6.4 Hz,
3
H, CH ), 3.17 (t, J= 5.1 Hz, 4H, 2CH ), 3.63 (t, J= 5.1 Hz, 2H, CH ), 3.66 (s, 3H, CH ),
2
2
2
3
.77-3.86 (m, 4H, 2CH ), 6.88-6.96 (m, 4H, Ar-H), 7.28-7.36 (m, 3H, Ar-H), 7.66-7.70 (m,
2
1
3
H, Ar-H), 8.22 (dd, J= 8.0Hz, 1.5 Hz, 1H, Ar-H), 10.63 (t, J= 5.7Hz, 1H, CONH); C NMR
(
125MHz, CDCl ): δ 29.13, 32.76, 35.02, 45.29, 49.36, 96.83, 114.17, 116.59, 120.49,
3
1
22.24, 125.44, 129.16, 133.65, 139.91, 150.85, 169.38, 171.11, 171.79. (ESI):m/z 435
+
[
M+H] ; HRMS: Calcd for C H O N : 435.20268, Found: 435.20198.
2
4
26
4
4
4
.9.3 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-(4-(4-(trifluoromethyl)phenyl)piperazin-1-
yl)propyl)-1,2-dihydroquinoline-3-carboxamide (8c):
1
2

ACCEPTED MANUSCRIPT
White solid (80% yield)) : m.p:134-137 ºC; IR (KBr, ν): 3101, 2924, 2855, 1633, 1574,
-
1 1
1
2
3
2
5
9
1
5
408, 1338, 1155, 1098, 1072,765 cm ; H NMR (500 MHz, CDCl ): δ 2.73 (t, J= 6.4 Hz,
3
H, CH ), 3.27 (t, J= 5.1 Hz, 4H, 2CH ), 3.64 (s, 3H, CH ), 3.65-3.68 (m, 2H, CH ), 3.78-
2
2
3
2
.84 (m, 4H, 2CH ), 6.90 (d, J= 8.6Hz, 2H, Ar-H), 7.28-7.35 (m, 2H, Ar-H), 7.44-7.49 (m,
2
H, Ar-H), 7.66-7.70 (m, 1H, Ar-H), 8.21 (dd, J= 8.1 Hz, 1.5Hz, 1H, Ar-H), 10.62 (t, J=
1
3
.4Hz, 1H, CONH); C NMR (75 MHz, CDCl ): δ29.16, 32.77, 35.01, 41.67, 45.50, 50.31,
3
6.83, 114.17, 116.06, 119.13, 122.26, 123.17, 125.45, 133.65, 162.50, 169.37, 171.10,
+
71.80; MS (ESI): m/z 503[M+H] ; HRMS: Calcd for C H O N F : 503.19007, Found:
25
25
4
4 3
03.19005.
4
.9.4 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-(piperidin-1-yl) propyl)-1,2-
dihydroquinoline-3-carboxamide (8d)
Pale yellow solid (78% yield)): m.p:148-152 C; IR (KBr, ν): 3422, 2926, 2853,
o
-1 1
1
633, 1567, 1445, 1411, 1337, 1252, 1015, 763 cm ; H NMR (500 MHz, CDCl ): δ 1.52-
3
1
.58 (m, 4H, 2CH ), 1.61-1.65 (m, 2H, CH ), 2.66 (t, J= 6.6 Hz, 2H, CH ), 3.39 (t, J= 5.3 Hz,
2
2
2
2
H, CH ), 3.59 (t, J= 5.4 Hz, 2H, CH ), 3.68 (s, 3H, CH ), 3.77 (q, J= 6.4Hz, 2H, CH ), 7.27-
2 2 3 2
7
.32 (m, 1H, Ar-H), 7.33-7.37 (m, 1H, Ar-H), 7.65-7.70 (m,1H, Ar-H), 8.21 (dd, J = 7.9 Hz,
1
3
1
.5Hz, 1H, Ar-H), 10.60 (t, J=6.5Hz, 1H, CONH); C NMR (75 MHz, CDCl ): δ25.42,
3
2
6.32, 29.64, 32.81, 35.08, 42.62, 46.36, 96.17, 114.14, 122.18, 125.44, 133.59, 162.50,
+
1
68.96, 171.81; MS (ESI): m/z 358[M+H] ; HRMS: Calcd for C H O N : 358.17613,
19
23
4
3
Found:358.17672.
4
.9.5 4-hydroxy-1-methyl-N-(3-(4-(4-nitrophenyl)piperazin-1-yl)-3-oxopropyl)-2-oxo-1,2-
dihydroquinoline-3-carboxamide (8e):
Red solid (77% yield)): m.p:169-173 C; IR (KBr, ν): 3249, 2925, 2854, 1725, 1633,
o
-1 1
1
595, 1307, 1112, 1020, 751cm ; H NMR (500 MHz, CDCl ): δ 2.74 (t, J= 6.4 Hz, 2H,
3
CH ), 3.42-3.49 (m, 4H, 2CH ), 3.65 (s, 3H, CH ), 3.66-3.71(m, 2H, CH ), 3.81 (q, J= 6.4
2
2
3
2
Hz, 2H, CH ), 3.85 (t, J= 5.4 Hz, 2H, CH ), 6.78-6.82 (m, 2H, Ar-H), 7.29-7.36 (m, 2H, Ar-
2
2
H), 7.67-7.71 (m,1H, Ar-H), 8.09-8.13 (m, 2H, Ar-H), 8.21 (dd, J=7.9Hz, 1.3Hz, 1H, Ar-H),
1
3
1
4
1
0.63 (t, J= 5.3Hz, 1H, CONH); C NMR (75 MHz, CDCl ): δ29.10, 32.75, 35.04, 41.21,
3
4.97, 48.01, 77.31, 76.99, 76.67, 96.81, 114.90, 114.17, 122.27, 125.43, 126.44, 133.70,
+
39.91, 152.81, 171.80, 162.48, 171.15, 169.49; MS (ESI): m/z 480[M+H] ; HRMS: Calcd
for C H O N : 480.18776, Found: 480.18926.
2
4
25
6
5
1
3

ACCEPTED MANUSCRIPT
4
.9.6 N-(3-(4-ethylpiperazin-1-yl)-3-oxopropyl)-4-hydroxy-1-methyl-2-oxo-1,2-
dihydroquinoline-3-carboxamide (8f):
Light brown solid (83% yield)): m.p:165-170 C; IR (KBr, ν): 3564, 3240, 2925,
o
-1 1
2
853, 1633, 1584, 1339, 1231, 1097, 956, 747 cm ; H NMR (300MHz, CDCl ): δ 1.03 (t,
3
J= 7.1Hz, 3H, CH ), 2.41-2.44 (m, 6H, 3CH ), 2.67 (t, J= 6.4 Hz, 2H, CH ), 3.48 (t, J= 4.8
3
2
2
Hz, 2H, CH ), 3.67-3-69 (m, 5H, CH CH ), 3.74-3.79 (m, 2H, CH ), 7.29-7.36 (m, 2H, Ar-
2
3,
2
2
H), 7.66-7.70 (m, 1H, Ar-H), 8.21 (dd, J = 7.9 Hz, 1.2 Hz, 1H, Ar-H), 10.62 (t, J= 5.1Hz, 1H,
1
3
CONH); C NMR (75 MHz, CDCl ): δ11.77, 29.58, 32.69, 34.95, 41.46, 45.23, 52.10,
3
5
3
2.72, 96.71, 114.09, 122.17, 125.36, 133.55, 139.83, 162.47, 169.14, 170.99;MS (ESI): m/z
+
87[M+H] ; HRMS: Calcd for C H O N : 387.20268, Found : 387.20369.
2
0
26
4
4
4
.9.7 N-(3-(4-benzylpiperazin-1-yl)-3-oxopropyl)-4-hydroxy-1-methyl-2-oxo-1,2-
dihydroquinoline-3-carboxamide (8g):
Brown solid (80% yield)): m.p:172-177 C; IR (KBr, ν): 3425, 3076, 2924, 2853,
o
-
1 1
1
2
638, 1568, 1458, 1336, 1214, 1082, 954, 751 cm ; H NMR (500 MHz, CDCl ): δ 2.41-
3
.45 (m, 4H, 2CH ), 2.63-2.68(m, 2H, CH ), 3.14-3.19 (m, 2H, CH ), 3.46 (t, J= 5.0 Hz, 2H,
2
2
2
CH ), 3.64-3.69 (m, 5H, CH , CH ), 3.76 (q, J=6.4 Hz, 2H, CH ), 7.29-7.37 (m, 7H, Ar-H),
2
3
2
2
7
.64-7.72 (m, 1H, Ar-H), 8.21(dd, J = 8.1 Hz, 1.3 Hz, 1H, Ar-H), 10.62 (t, J=5.9Hz, 1H,
1
3
CONH); C NMR (75 MHz, CDCl ): δ29.15, 34.98, 45.24, 50.10, 52.59, 62.78, 96.65,
3
1
14.16, 122.24, 125.43, 127.25, 128.27, 129.11, 133.62, 137.33, 160.74, 169.22, 171.03; MS
+
(
ESI): m/z 449[M+H] ; HRMS: Calcd for C H O N : 449.21833, Found : 449.21891.
2
5
28
4
4
4
1
.9.8 4-hydroxy-N-(3-(4-(2-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)-1-methyl-2-oxo-
,2-dihydroquinoline-3-carboxamide (8h):
o
Light brownsolid (76% yield): m.p:162-164 C; IR (KBr, ν): 3251, 2925, 2855, 1629,
-
1 1
1
2
3
7
1
3
1
589, 1499, 1335,1273, 1024, 751 cm ; H NMR (300 MHz, CDCl ): δ 2.73(t, J= 6.4 Hz,
3
H, CH ), 2.99-3.08 (m, 4H, 2CH ), 3.61-3.65 (m, 2H, CH ), 3.66 (s, 3H, CH ), 3.76-
2
2
2
3
.81(m, 2H, CH ), 3.83-3.86 (m, 2H, CH ), 3.87 (s, 3H, CH ), 6.85-6.91 (m, 3H, Ar-H), 6.98-
2
2
3
.05 (m, 1H, Ar-H), 7.28-7.36 (m, 2H, Ar-H), 7.64-7.77 (m, 1H, Ar-H), 8.20 (dd, J = 7.9 Hz,
1
3
.1 Hz, 1H, Ar-H), 10.63 (t, J=5.6 Hz, 1H, CONH); C NMR (125 MHz, CDCl ): δ 29.15,
3
2.76, 35.08, 45.65, 50.53, 55.39, 96.86, 111.20, 114.16, 118.36, 120.96, 122.24, 123.50,
+
25.46, 133.63, 140.57, 152.18, 169.31, 171.11, 171.82; MS(ESI): m/z 465[M+H] ; HRMS:
Calcd for C H O N : 465.21325, Found: 465.21294.
2
5
28
5
4
1
4

ACCEPTED MANUSCRIPT
4
.9.9 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-thiomorpholinopropyl)-1,2-
dihydroquinoline-3-carboxamide (8i):
Brown liquid (68% yield)): IR (KBr, ν): 3247, 2925, 2814, 1634, 1567, 1454, 1209,
-
1 1
1
6
7
145, 1004, 747cm ; H NMR (500 MHz, CDCl ):δ 2.31-2.54 (m, 4H, 2CH ), 2.67 (t, J =
3
2
.4 Hz, 2H, CH ), 3.65-3.67 (m, 4H, 2CH ), 3.68 (s, 3H, CH ), 3.78 (q, J = 6.25, 2H, CH ),
2
2
3
2
.28-7.41 (m, 2H, Ar-H), 7.59-7.73 (m, 1H, Ar-H), 8.18-8.30 (m, 1H, Ar-H), 10.62 (t, J = 5.3
1
3
Hz,1H, CONH); C NMR (125 MHz, CDCl ): δ26.38, 29.70, 32.87, 35.14, 42.67, 96.23,
3
+
1
14.20, 122.24, 125.50, 133.64, 162.56, 169.02, 171.87; MS (ESI): m/z 376[M+H] ; HRMS:
Calcd for C H O N S: 376.13255, Found: 376.13316.
1
8
21
4
3
4
.9.10 4-hydroxy-1-methyl-N-(3-(4-methylpiperazin-1-yl)-3-oxopropyl)-2-oxo-1,2-
dihydroquinoline-3-carboxamide (8j):
Light brown liquid (79% yield); IR (KBr, ν): 3235, 2930, 2796, 1632, 1563, 1504,
-
1 1
1
2
445, 1337, 1144, 1002, 762 cm ; H NMR (500 MHz,CDCl ): δ2.29 (s, 3H, CH ), 2.36-
3 3
.41 (m, 4H, 2CH ), 2.67 (t, J = 6.4 Hz, 2H, CH ), 3.48 (t, J = 5.0 Hz, 2H, CH ), 3.64-3.70
2
2
2
(
m, 5H, CH CH ), 3.75-3.79 (m, 2H, CH ), 7.28-7.36 (m, 2H, Ar-H), 7.68 (t, J = 7.6 Hz, 1H,
3, 2 2
1
3
Ar-H), 8.19-8.22(m, 1H, Ar-H), 10.60 (t, J= 6.1 Hz, 1H, CONH); C NMR (75 MHz,
CDCl ): δ29.15, 32.76, 35.02, 96.84, 114.16, 122.25, 125.46, 133.62, 139.93, 162.52, 169.21,
3
+
1
3
71.08; MS (ESI): m/z373[M+H] ; HRMS: Calcd for C H O N : 373.18703, Found :
1
9
24
4
4
73.18598.
4
1
.9.11 4-hydroxy-N-(3-(4-(3-methoxyphenyl)piperazin-1-yl)-3-oxopropyl)-1-methyl-2-oxo-
,2-dihydroquinoline-3-carboxamide (8k):
o
Light brown solid (74% yield) : m.p:165-168 C; IR (KBr, ν): 3446, 3084, 2923,
-1 1
2
856, 1633, 1571, 1496, 1438, 1335, 1227, 1017, 927, 763, 695 cm ; H NMR (500 MHz,
CDCl ): δ2.72 (t,J= 6.4 Hz, 2H, CH ), 3.14-3.18 (m, 4H, 2CH ), 3.60-3.64 (m, 2H,
3
2
2
CH ),3.66 (s, 3H, CH ), 3.78 (s, 3H, CH ), 3.79-3.81 (m, 4H, 2CH ), 6.43-6.48 (m, 2H, Ar-
2
3
3
2
H), 7.14-7.22 (m, 2H, Ar-H), 7.27-7.36 (m, 2H, Ar-H), 7.66-7.71 (m, 1H, Ar-H), 8.21(dd, J =
1
3
8
.0 Hz, 1.5 Hz, 1H, Ar-H), 10.62 (t, J= 5.8Hz, 1H, CONH); C NMR (75 MHz, CDCl ):
3
δ32.79, 35.05, 41.48, 45.25, 49.30, 55.18, 96.85, 103.06, 105.52, 109.26, 114.18, 122.27,
+
1
25.48, 129.89, 133.66, 160.56, 169.42, 171.84; MS (ESI): m/z 465 [M+H] ; HRMS: Cacld
for C H O N : 465.21325, Found: 465.21077.
2
5
28
5
4
1
5

ACCEPTED MANUSCRIPT
4
.9.12 N-(3-(4-benzylpiperidin-1-yl)-3-oxopropyl)-4-hydroxy-1-methyl-2-oxo-1,2-
dihydroquinoline-3-carboxamide (8l):
Light yellow solid (77% yield);m.p: 154-158 C; IR (KBr, ν): 3247, 2925, 2854,
o
-
1 1
1
1
3
1
8
724, 1638, 1284, 1110,746 cm ; H NMR (500 MHz,CDCl ): δ1.14-1.26 (m, 4H, 2CH ),
3 2
.72-1.77 (m,1H, CH), 2.52-2.58 (m, 2H, CH ), 2.63-2.66 (m, 2H, CH ), 3.66 (s, 3H, CH ),
2
2
3
.70-3.83 (m, 6H, 3CH ), 7.08-7.12 (m, 2H, Ar-H), 7.17-7.21 (m,1H, Ar-H), 7.23-7.25 (m,
2
H, Ar-H), 7.27-7.31 (m, 2H, Ar-H), 7.33-7.36 (m, 1H, Ar-H), 7.62-7.70 (m, 1H, Ar-H),
1
3
.18-8.22 (m, 1H, Ar-H), 10.59 (t, J = 5.8 Hz, 1H, CONH); C NMR (75 MHz, CDCl3):
δ29.12, 32.38, 38.16, 41.97, 42.89, 45.63, 96.83, 114.13, 125.42, 125.97, 128.22, 128.99,
+
1
33.59, 139.87,162.48, 169.00, 171.02, 171.80; MS (ESI): m/z 449 [M+H] ; HRMS: Calcd
for C H N O : 448.22068, Found: 448.22275.
2
6
30
3
4
Acknowledgements
The authors gratefully acknowledge the financial support through the project: DST-
SERB/EMEQ-078/2013andthe University grant commission (UGC), New Delhi for the
award of fellowship to SB.
References
[
1] A.K. El-Damasy, S.H. Seo, N. C. Cho, S. B. Kang, A. N. Pae, K. S. Kim, G. Keum.
Design, synthesis, in-vitro antiproliferative activity and kinase profile of new
picolinamide based 2-amido and ureido quinoline derivatives, Eur. J. Med. Chem. 101
(2015) 754-768.
[
2] V. Spano, B. Parrino, A.Carbone, A.Montalbano ,A. Salvador , P. Brun , D. Vedaldi , P.
Diana, G. Cirrincione, P. Barraja,Pyrazolo[3,4-h] quinolines promising photosensitizing
agentsin the treatment of cancer, Eur. J. Med. Chem. 102 (2015) 334-351.
[
3] I. Briguglio, R. Loddo, E. Laurini, M. Fermeglia, S. Piras, P. Corona, P. Giunchedi, E.
Gavini, G. Sanna, G. Giliberti,C. Ibba, P. Farci, P. L. Colla, S. Pricl,A. Carta.Synthesis,
cytotoxicity and antiviral evaluation of new series of imidazo[4,5-g]quinoline and
pyrido[2,3-g] quinoxalinone derivatives,Eur. J. Med. Chem. 105 (2015) 63-79.
4] T. Kalland, Effects of the Immunomodulator Ls 2616 on Growth and Metastasis of the
Murine B16-F10 Melanoma, Cancer Res. 46 (1986) 3018-3022.
[
[
5] T. Ichikawa, J. C. Lamba, P. I. Christensson, B. Hartley-Asp. The Antitumor Effects of
The Quinoline-3-Carboxamide Linomide on Dunning R-3327 Rat Prostatic Cancers,
1
6

ACCEPTED MANUSCRIPT
Cancer Res. 52 (1992) 3022-3028.
[
6] A. Tarkowski, K. Gunnarsson, L-A, Nilson, L. Lindholm, T. Stalhandske. Succesfull
Treatment of Autoimmunity in MRL/1 mice with LS-2616, a new immunomodulator,
Arthr.Rheum.29 (1986) 1405. T. Kalland, Effects of the Immunomodulator Ls 2616
on Growth and Metastasis of the Murine B16-F10 Melanoma, Cancer Res. 46 (1986)
3
018-3022.
[
[
7] A. Tarkowski, K. Gunnarsson, T. Stalhandske, Effects of LS-2616 administration upon
The autoimmune disease (NZBXNZW) F1 hybrid mice, Immunology. 59 (1986) 589-
5
94.
8] D. M. Karussis, D. Lehmann, S. Salvin, U. Vourka-Karussis, R. Mizrachi-Koll, H.
Ovadia, T. Kalland, O. Abramsky, Treatment of Chronic-relapsing experimental
autoimmune encephalomyelitics with the synthetic immunomodulator linomide. Proc.
Natl. Acad. Sci. U.S.A. 90 (1993) 6400-6404.
[
9] D. M. Karussis, D. Lehmann, S. Salvin, U. Vourka-Karussis, R. Mizrachi-Koll, H.
Ovadia; D. Ben-Num, T. Kalland, O. Abramsky, Inhibition of acute, experimental
autoimmune encephalomyelitis by the synthetic immunomodulator linomide. Ann.
Neurol. 34 (1993) 654-660.
[
[
[
10] O. Andersen, J. Lycke, P. O. Tollesson, A. Svenningsson, B. Runmarker, A. S. Linde,
M. Astrom, P. Gjorstrup, S. Ekholm,Linomide reduces the rate of active lesions in
relapsing-remitting multiple sclerosis,Mult. Scler.1(1996) 348.
11] O. Andersen, J. Lycke, P. O. Tollesson,. A. Svenningsson, B. Runmarker, A. S. Linde,
M. Astrom, P. Gjorstrup, S. Ekholm, Linomide reduces the rate of active lesions in
relapsing-remitting multiple sclerosis, Neurology 47 (1996) 895-900.
12] D. M. Karussis, Z. Meiner, D. Lehmann, J. M. Gomori, A. Schwarz, A. Linde,
O. Abramsky, Treatment of secondary progressive multiple sclerosis with the
immunomodulator linomide: a double-blind, placebo controlled pilot study with
monthly magnetic resonance imaging evaluation, Neurology, 47 (1996) 341-346.
13] J.T Isaacs, R. Pili, D.Z. Qian, S.L. Dalrymple, J.B. Garrison, N .Kyprianou,A. Björk,
A. Olsson, T. Leanderson, Identification of ABR-215050 as lead secondgeneration
quinoline-3-carboxamide anti-angiogenic agent for the treatment of prostate cancer,
Prostate. 66 (2006) 1768–78.
[
[
14] J.T. Isaacs, L. Antony, S.L. Dalrymple, W.N. Brennen, S.Gerber, H.Hammers, et al,
Tasquinimod is an allosteric modulator of HDAC4 survival signaling within the
compromised cancer microenvironment, Cancer Res. 73 (2013) 1386-1399.
1
7

ACCEPTED MANUSCRIPT
[
`
15] J. H. Noseworthy, J. S. Wolinsky, F. D.Lublin, Linomide in relapsing and
secondary progressive MS: part I: trial design and clinical results. North American
Linomide investigators,Neurology,54 (2000) 1726-1733.
[
16] S. Jönsson , G. Andersson, T. Fex , T. Fristedt, G. Hedlund , K. Jansson, L. Abramo,
I. Fritzson, O.Pekarski,A. Runström, H.Sandin, I.Thuvesson, A. Björk, Synthesis
and biological evaluation of new 1,2-dihydro-4-hydroxy-2-oxo-3-quinoline
carboxamides for treatment of autoimmune disorders: structure-activity relationship, J.
Med. Chem. 47 (2004) 2075-2088.
[
[
17] C. Polman, F. Barkhof, M. Sandberg-Wollheim, A. Linde, O. Nordle, T. Nederman,
Treatment with laquinimod reduces development of active MRI lesions in relapsing
MS, Neurology. 64 (2005) 987-991.
18] R. J. Kerns, M. J. Rybak, G. W.Kaatz, F.Vaka. R. Cha, R.G. Grucz, V.U. Diwadkar,
Structural features of piperazinyl-linked ciprofloxacin dimers required foractivity
against drug-resistant strains of Staphylococcus aureus, Bioorg.Med. Chem.Lett.
1
3(2003)2109-2112.
[
[
[
[
[
19] A. Ryckebusch, R. Deprez-Poulain, L. Maes, M.A. Debreu-Fontaine, E. Mouray,
P. Grellier, C. Sergheraert, Synthesis and invitro and invivo antimalarial activity of N-
(
7-Chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine derivatives, J. Med. Chem. 46
2003) 542-557.
(
20] U. Ram Shankar, S. Neelima, J. Sanjay, K. Nawal, C. Ramesh, A. Sudershan,
Optically Active antifungal azoles: Synthesis and antifungal activity of (2R,3S)-2-(2,4-
difluorophenyl)-3-(5-{2[4-aryl-piperazin-1-yl]-ethyl}-tetrazoll-2-yl/1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol, Biorg.Med. Chem. 12 (2004)2225-2238.
21] M.X. Dong , L. Lu , H. Li , X. Wang , S. Jiang , Q.Y Dai ,Design, synthesis, and
biological activity of novel 1,4-disubstituted piperidine/piperazine derivatives as
CCR5 antagonist-based HIV-1 entry inhibitors, Bioorg. Med. Chem. Lett
2012) 3284-3286.
.
22(9)
(
22] A. Foroumadi, S. Emami, A. Hassanzadeh, M. Rajaee, K. Sokhanvar, M. H.
Moshafi, A. Shafiee, Synthesis and antibacterial activity of N-(5-benzylthio-1, 3, 4-
thiadiazol-2-yl) and N-(5-benzyl sulfonyl-1, 3, 4-thiadiazol-2-yl) piperazinyl
quinolone derivatives, Biorg. Med. Chem. Lett. 15(20) (2005) 4488-4492.
23] G. D. Abuo-Rehma, H. A. Sarhan, G. F. M. Gad, Design, synthesis, antibacterial
activity and physicochemical parameters of novel N-4-piperazinyl derivatives of
1
8

ACCEPTED MANUSCRIPT
Norfloxacin
,
Bioorg. Med. Chem. 17 (2009)3879-3886.
[
[
24] S. Wang, X. D. Jia, M. L. Liu, Y. Lu, H. Y. Guo, Synthesis, antimycobacterial and
antibacterial activity of ciprofloxacin derivatives containing a N-substituted benzyl
moiety, Bioorg. Med. Chem. Lett. 22(18) (2012) 5971-5975.
25] L. Nagarapu, H. K. Gaikwad, K. Sirikonda, J. Mateti, R. Bantu, P. S. Raghu, K. M.
Mahduri, S. V. Kalivendi. Synthesis and cytotoxicity evaluation of 1-[3-(9H-
carbazol-4-yloxy)-2-hydroxypropyl]-3-aryl-1H-pyrazole-5-carboxylic acid derivatives,
Eur. J. Med. Chem. 45 (2010) 4720-4725; b) L.Nagarapu, J. Mateti, H. K. Gaikwad, R.
Bantu, M. Sheebarani, N. J. P. Shubhasini, Synthesis and anti-inflammotory activity of
some novel 3-phenyl-N-[3-(4-phenylpiperazin-1yl) propyl] 1H-pyrazole-5-carboxamide
derivatives, Bioorg Med. Chem. Lett. 21 (2011) 4138-4140; c) L. Nagarapu, S.
Vanaparthi, R. Bantu, C. G. Kumar, Synthesis of noval benzo[4,5]thiazolo[1,2-
a]pyrimidine-3-carboxylate derivatives and biological evaluation as potential anticancer
agents, Eur. J. Med. Chem. 69 (2013)817-822.
[
[
26] Michael Goldbrunner, Günther Loidl, Thomas Polossek, Albrecht Mannschreck,
and Erwin von Angerer, Inhibition of Tubulin Polymerization by 5,6-Dihydroindolo[2,1-
a]isoquinoline Derivatives, Journal of Medicinal Chemistry,40 (1997) 3524-3533.
27] Yu-Hsun Chang, Mei-Hua Hsu, Sheng-Hung Wang, Li-Jiau Huang, Keduo Qian, Susan
L. Morris-Natschke, Ernest Hamel, Sheng-Chu Kuo and Kuo-Hsiung Lee, Design and
synthesis of 2-(3-benzo[b]thienyl)-6,7-methylenedioxyquinolin-4- one analogues as
potent antitumor agents that inhibit tubulin assembly, Journal of Medicinal Chemistry,
52(2009) 4883-4891.
[
28] Romeo Romagnoli, Pier Giovanni Baraldi, Maria Kimatrai Salvador, Filippo Prencipe,
Carlota Lopez-Cara, Santiago Schiaffino Ortega, Andrea Brancale, Ernest Hamel,
Ignazio Castagliuolo, Stefania Mitola, Roberto Ronca, Roberta Bortolozzi, Elena Porcù,
Giuseppe Basso, and Giampietro Viola, Design, Synthesis, in Vitro and in Vivo
Anticancer and Antiangiogenic Activity of Novel 3-Arylamino Benzofuran Derivatives
Targeting the Colchicine Site on Tubulin, Journal of Medicinal Chemistry 58 (2015)
3
209-3222.
[
29] B. Keerthana, J. Swathi Reddy, T. Saritha Jyostna, S. Sree Kanth, M. Vijjulatha, Design,
synthesis, molecular docking and biological evaluation of new dithiocarbamates
substituted benzimidazole and chalcones as possible chemotherapeutic agents, Biorg.
Med. Chem Lett. 22 (2012), 3274-3277.
1
9

ACCEPTED MANUSCRIPT
[
[
30] Schrödinger, LLC, Glide, Version 5.6 (2010), New York, NY.
31] R.B. Ravelli, B. Gigant, P.A. Curmi, I. Jourdain, S. Lachkar, A. Sobel,
Knossow, M, Insight into tubulin regulation from a complex with colchicines and a
stathmin-like domain, Nature. 428 (2004) 198-202.
[
[
32] Ligprep 2.0, Schrodinger, LLC, New York, NY, USA, 2010.
33] G. A. Kaminski, R. A. Friesner, J. Tirado-Rives, and W. L. Jorgensen, Evaluation and
reparametrization of the OPLSAA force field for proteins via comparison with accurate
quantum chemical calculations on peptides, Journal of Physical Chemistry B, vol. 105,
no. 28, (2001) 6474–6487.
Supplementary data
1
13
Copies of the H, C NMR, ESI-MS and HRMS spectra for some of the important
compounds.
Figure Captions:
Figure1: Structure of Roquinimex and roquinimex based under clinical trial cancer therapy
agents.
Figure2: Representative examples of biologically active piperazinyl substituted quinoline
derivatives.
Figure 3: Design strategy for 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-piperazinyl) propyl)-1,
2
-dihydroquinoline-3-carboxamide hybrids.
Figure 4: a,b,c represents the dock poses of Combretastatin, 8d and 8e molecules in the
tubulin active site.
Figure 5:Scatter plots of Experimental pGI VS Dock score values,
5
0
Activity 1 (PANC1)(a), Activity 2 (HeLa)(b) and Activity 3 (MDA-MB-231)(c).
Scheme1.Synthesis of4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid4
Scheme2.Synthesis of 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-(piperazinyl) propyl)-1,2-
dihydroquinoline-3-carboxamide hybrids 8(a-l).
Table1
:4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-(piperazinyl) propyl)-1, 2-dihydroquinoline-
3
-carboxamide hybrids 8(a-l).
Table 2
Table 3
Table 4
:
:
:
GI values of the tested compounds against different human cancer cell lines.
50
Molecules with their activities and dock scores.
Pharmacokinetic properties of molecules
2
0

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Table 1: 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-piperazinyl) propyl)-1,2-dihydroquinoline-3-
carboxamide hybrids 8(a-l).
Entry
2o amine
Product
yielda
Compound
OH O
O
OH O
O
HN
N
H
OH
N
F
N
N
a
82
H
N
F
N
CH3
O
N
CH3
O
OH O
O
HN
b
6
N
H
N
N
N
N
8
4
N
N
O
CH3
OH O
O
HN
c
6
6
N
H
80
N
N
CH3
O
CF3
CF3
OH O
O
O
d
HN
HN
N
H
N
N
7
8
N
CH3
OH O
O
N
H
e
f
6
6
77
83
N
N
N
N
CH3
O
NO2
NO2
OH O
O
O
O
HN
N
H
N
N
N
CH
3
N
CH
3
N
CH
OH O
O
3
HN
HN
N
H
g
h
6
6
N
N
N
N
8
0
6
N
CH3
O
OH O
N
H
7
N
O
H CO
CH3
3
H CO
3
OH O
O
N
N
i
j
6
6
HN
6
8
H
S
S
N
CH3
O
O
OH O
HN
N
N
79
N
H
CH3
N
N
O
CH3
CH3
2
1

ACCEPTED MANUSCRIPT
a
Percentage yields of isolated products
a
Table 2: (GI ) values of the tested compounds against three human cancer cell lines.
50
S.No
PANC 1
2.5±0.09
HeLa
MDA-MB-231
1.7±0.03
1.62±0.08
5.4±0.17
1.4±0.05
0.3±0.02
1.32±0.16
0.51±0.01
1.2±0.06
2.25±0.03
0.59±0.02
1.3±0.05
0.71±0.01
<0.01
8
8
a
0.96±0.03
3.9±0.09
3.7±0.06
0.82±0.03
0.94±0.02
0.88±0.04
0.76±0.02
0.74±0.01
3.0±0.19
1.16±0.04
0.73±0.02
1.4±0.06
<0.01
b
1.57±0.04
2.55±0.04
0.15±0.02
0.42±0.01
0.48±0.03
0.95±0.01
0.7±0.01
8
c
8
d
8
8
8
e
f
g
8
h
8
i
1.91±0.06
1.66±0.04
0.51±0.01
0.68±0.03
<0.01
8
j
8
k
8
l
Nocodazole
Combretastatin
0.059±0.002
0.083±0.001
0.09±0.01
a
GI : 50% Growth inhibition, concentration of drug (in ꢀM) resulting in a 50% reduction in
5
0
net protein increase compared with control cells.
b
Positive controls.
2
2

ACCEPTED MANUSCRIPT
Table 3: Molecules with their activities and dock scores.
Compd.
PANC 1
HeLa
MDA-MB-231
Dock
score(XP)
GI50
pGI50
5.6020
5.8041
5.5934
6.8239
6.3767
6.3187
6.0222
6.1549
5.7189
5.7798
6.2924
6.1674
GI50
pGI50
6.0177
5.4089
5.4317
6.0861
6.0268
6.0555
6.1191
6.1307
5.5228
5.9355
6.1366
5.8538
GI50
pGI50
8
8
8
8
8
a
b
c
2.5±0.09
1.57±0.04
2.55±0.04
0.15±0.02
0.42±0.01
0.48±0.03
0.95±0.01
0.7±0.01
1.91±0.06
1.66±0.04
0.51±0.01
0.68±0.03
0.96±0.03
3.9±0.09
3.7±0.06
0.82±0.03
0.94±0.02
0.88±0.04
0.76±0.02
0.74±0.01
3.0±0.19
1.16±0.04
0.73±0.02
1.4±0.06
1.7±0.03
1.62±0.08
5.4±0.17
1.4±0.05
0.3±0.02
1.32±0.16
0.51±0.01
1.2±0.06
2.25±0.03
0.59±0.02
1.3±0.05
0.71±0.01
0.09±0.01
5.7695
5.7904
5.2676
5.8538
6.5228
5.8794
6.2924
5.9208
5.6478
6.2291
5.8860
6.1487
7.0457
-4.854
-4.041
-4.508
-5.734
-5.709
-4.161
-5.085
-5.376
-5.410
-2.649
-5.1
d
e
8
f
8
8
8
8
8
8
g
h
i
j
k
l
-4.073
-6.793
Combr 0.059±0.002 7.2291
etastatin
0.083±0.001 7.0809
Nocodaz <0.01
ole
-
<0.01
-
<0.01
-
-5.753
Table4: Pharmacokinetic properties of molecules.
a
b
c
d
e
Compd. QPlogPo/w QPlogS QPlogBB QPPCaco QPPMDCK %Human oral
f
absorption
8
8
8
a
b
c
3.792
4.128
4.723
-4.077
-4.803
-5.556
-0.729
-0.611
-0.57
454.113
843.169
462.949
564.204
796.743
1869.451
96.704
100
100
2
3

ACCEPTED MANUSCRIPT
8
e
f
d
3.05
3.55
-4.106
-6.14
-0.837
-2.053
-0.576
-0.673
-1.08
548.13
93.559
464.888
57.006
93.827
83.012
77.902
87.241
100
8
8
2.168
3.583
4.294
3.031
1.819
4.264
5.21
-2.723
-4.433
-5.806
-4.027
-1.47
137.328
157.245
464.425
631.118
243.423
454.509
771.018
2087.011
98.524
8
g
113.883
406.092
928.994
216.776
402.52
8
h
8i
-0.657
-0.106
-1.082
-0.899
-0.654
94.811
80.308
100
8
8
j
k
-5.747
-6.391
-3.842
8
l
625.161
1095.768
96.168
100
Combre 3.327
tastatin
a
Molecules.
b
Predicted octanol/water partition coefficient log P (Acceptable range-2.0 to 6.5)
Predicted aqueous solubility’s in mol/L (Acceptable range -6.5 to 0.5)
Predicted BBB permeability (Acceptable range-3 to 1.2)
Predicted Caco cell permeability in nm/s (Acceptable range :< 25 is poor and >500 is great)
c
d
e
f
Predicted apparent MDCK cell permeability in nm/s (Acceptable range in nm/s (Acceptable range : <
2
5 is poor and>500 is great)
g
Percentage of human oral absorption (Acceptable range: <25 is poor and >80% is high).
Figure 1.Quinolines containing biologically active molecules
2
4

ACCEPTED MANUSCRIPT
Figure 2:Representative examples of biologically active piperazinyl substituted quinoline
derivatives.
Figure 3:Design strategy for new 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-piperazinyl)
propyl)-1, 2-dihydroquinoline-3-carboxamide hybrids.
o
Reagents and conditions: i) CH I, NaH DMF,5 h ii) Diethyl malonate, NaH, DMF, 120 C,
3
,
o
reflux, 12 h iii) CH CO H, HCl, CH CH OH, 60 C, 4 h, reflux.
3
2
3
2
Scheme 1:Synthesis of4-hydroxy-1-methyl-2-oxo-1, 2-dihydroquinoline-3-carboxylicacid (4).
2
5

ACCEPTED MANUSCRIPT
X = a) N-Ph-2F; b) N-Ph; c) N-Ph-4CF ;d) CH ; e) N-Ph-4NO ;f) N-CH CH ;g) N-
3
2
2
2
3
CH -Ph; h) N-CH -2OCH i) S; j) N-CH ; k) N-CH -2OCH l) CH -CH - Ph
2
2
3;
3
2
3;
2
2
Reagents and conditions: i) β-Alanine ethyl ester HCl. EDC. HCl, HOBT, ET N, DCM, rt, 16 h
3
o
ii) 1N KOH, ethanol, 80 C, reflux, 4 h iii) EDC. HCl, HOBT, DCM, rt, 24 h.
Scheme 2:Synthesis of 4-hydroxy-1-methyl-2-oxo-N-(3-oxo-3-piperazinyl) propyl)-1,2-
dihydroquinoline-3-carboxamide hybrids (8a-l).
2
6

ACCEPTED MANUSCRIPT
(a)
(b)
(c)
Figure 4: a,b,c represents the dock poses of Combretastatin, 8d and 8e molecules in the
tubulin active site.
2
7

ACCEPTED MANUSCRIPT
SCATTER PLOTS:
Scatter plot of Expt.
Activity Vs Dock score
7
6
5
.5
7
.5
6
.5
5
-
2
-4
-6
-8
Dock score
(a)
Scatter plot of Expt.
Activity Vs Dock score
8
7
6
5
-
2
-4
-6
-8
Dock score
(b)
Scatter plot of Expt.
Activity Vs Dock score
7
.5
7
6
.5
6
5
.5
5
-
3
-5
-7
Dock score
(c)
Figure 5: Scatter plots of Experimental pGI VS Dock score values
5
0
Activity 1 (PANC1) (a), Activity 2 (HeLa) (b) and Activity 3 (MDA-MB-231) (c).
2
8