Sulphonamido-quinoxalines: Search for anticancer agent

Article abstract of DOI:10.1016/j.ejmech.2013.04.028

A series of new sulphonamido-quinoxaline derivatives 3 (a-p) have been prepared which are structurally similar to the High Throughput Screening (HTS) hit identified by Porter and collaborator. The newly synthesized compounds 3b, 3c, 3f, 3i, 3j, 3l, 3n and 3o were further evaluated in the National Cancer Institute for in vitro cytotoxicity assay among them compound 3l showed highest activity against Leukemia RPMI-8226 cell lines (GI₅₀: 1.11 μM) as compared to other tested compounds. It is to be noted that compound 3l shows significant activity (GI₅₀: 1.11 μM) compared to the High Throughput Screening (HTS) hit identified by Porter and collaborator (IC ₅₀ = 1.3 μM). Further docking study confirms the c-Met kinase inhibitory mechanism of the synthesized compounds.

Full text of DOI:10.1016/j.ejmech.2013.04.028

European Journal of Medicinal Chemistry 65 (2013) 168e186
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Sulphonamido-quinoxalines: Search for anticancer agent
,
a
Rahul Ingle a, Rajendra Marathe b, Dipak Magar c, Harun M. Patel a , Sanjay J. Surana
*
a Department of Pharmaceutical Chemistry, R.C. Patel College of Pharmacy, Shirpur, Dhule 425405, Maharashtra, India
b Department of Pharmaceutical Chemistry, Government College of Pharmacy, Amravati 424605, Maharashtra, India
c Department of Pharmaceutical Chemistry, Dr. Bhanuben Nanawati College of Pharmacy, Mumbai 400056, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new sulphonamido-quinoxaline derivatives 3(aep) have been prepared which are structurally
similar to the High Throughput Screening (HTS) hit identified by Porter and collaborator. The newly
synthesized compounds 3b, 3c, 3f, 3i, 3j, 3l, 3n and 3o were further evaluated in the National Cancer
Institute for in vitro cytotoxicity assay among them compound 3l showed highest activity against Leu-
Received 16 February 2013
Received in revised form
12 April 2013
Accepted 15 April 2013
Available online 24 April 2013
kemia RPMI-8226 cell lines (GI50: 1.11
compound 3l shows significant activity (GI50: 1.11
(HTS) hit identified by Porter and collaborator (IC50 ¼ 1.3
kinase inhibitory mechanism of the synthesized compounds.
Ó 2013 Elsevier Masson SAS. All rights reserved.
m
M) as compared to other tested compounds. It is to be noted that
M) compared to the High Throughput Screening
M). Further docking study confirms the c-Met
m
Dedicated to my late mother Ms. Ajija Patel,
who was the source of inspiration for me.
m
Keywords:
Synthesis sulphonamido-quinoxaline
Anticancer
Docking
Lipinski’s rule
1. Introduction
generate biological responses in their interaction with several bio-
logical targets. They show antiviral [15], herbicidal [16] and anti-
inflammatory action [17]. Recent investigations revealed the phar-
macological potential of quinoxalines as anticancer agents [18] and
numerous theoretical studies were performed on quinoxaline and
its derivatives, in order to find new antineoplastic compounds.
Porter and collaborator recent investigation pointed out the
quinoxaline scaffold as a template to the design of inhibitors of c-
Met kinase [19]. In the course of one high throughput screening
campaign, they selected one quinoxaline derivative as the most
promising structure to inhibit selectively the ATP binding site of c-
Members of the receptor tyrosine kinase (RTK) family are
attractive targets for cancer therapy as inhibition can disrupt
signaling pathways that mediate tumor formation and growth [1e
3]. c-Met kinase is a member of this family that, together with its
ligand, hepatocyte growth factor (HGF) or scatter factor (SF), is
important for normal mammalian development. However, c-Met
has been shown to be deregulated and associated with high tumor
grade and poor prognosis in a number of human cancers [4,5]. c-Met
can become activated by a variety of mechanisms, including gene
amplification and mutation inducing motility, invasiveness and
tumourgenicity into the transformed cells [6e10]. Activation leads
to receptor dimerization and recruitment of several SH2 domain
containing signal transducers that activate a number of pathways
including the Raf-Mek-Erk and PI3k-Akt cascades [9,11e14]. Tar-
geting the ATP binding site of c-Met is a popular strategy for inhi-
bition of the kinase, many small molecules selectively targeting the
ATP binding site of c-Met kinase have been identified and exerted
significant therapeutic effects in treating human cancers clinically
[9,12,13,15,16]. Quinoxalines and quinoxalinones are attractive
chemical candidates in medicinal chemistry due to their capacity to
Met (IC50 ¼ 1.3
mM in the c-Met biochemical assay) [19]. After that,
they successfully optimized the activity of the original structure by
preparing and testing a set of quinoxalines. Following Porter’s
research, we decided to contribute in the analysis of the interaction
of quinoxalines with c-Met kinase.
Hence in continuation of our efforts on the design and syn-
thesis of novel anticancer agents [20e25] and keeping in mind the
medicinal importance of quinoxaline moiety, in the present study
we synthesized and in vitro evaluated quinoxalines at National
Cancer Institute (NCI-USA) for antitumor activity. We have also
tried to dock the synthesized compounds with the crystal struc-
ture of c-Met kinase (PDB: 2wgj) to explore the possible anti-
cancer mechanism of our compounds and similarly further
analyzed the synthesized compounds for Lipinski’s rule of five to
* Corresponding author. Tel.: þ91 8806621544; fax: þ91 1881263655.
E-mail address: hpatel_38@yahoo.com (H.M. Patel).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.028

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
169
evaluate drug likeness and established in silico ADME parameters
using QikProp.
accommodate into the hydrophobic pocket of c-Met kinase; there-
fore we introduced phenyl ring at 2nd and 3rd position of qui-
noxaline to increase hydrophobic interaction with receptor c-Met
kinase [19].
2. Rationale and designing
On the other hand PF-2341066 (crizotinib) has been identified as
an ATP-competitive c-Met inhibitor [13,26]. We analyzed the cri-
zotinib structure deposited in PDB (PDBcode: 2wgj) and identified
that the 2-aminopyridine (crizotinib) bound to the hinge region of c-
Met and interact with the amidal NH of Met-1160 via nitrogen of
pyridine forming hydrogen-bonding interaction. Furthermore, we
found that 2,6-di-chloro-3-fluorophenyl ring formed a hydrophobic
interaction with c-Met kinase (Fig. 3), these hydrophobic in-
teractions may contribute to the selective inhibition of c-Met kinase.
While exploring the scaffold of c-Met selective inhibitor, we
hypothesized that the hydrophobic interactions were essential and
the binding affinity enhanced byadding the hydrophobic space filler
In this study, we present a new sub-family of compounds con-
taining 2,3,6-trisubstituted quinoxalines as c-Met kinase inhibitors.
We have designed the ligands which are structurally similar to the
High Throughput Screening (HTS) hit identified by Porter and
collaborator as shown in Figs. 1e4 [19]. Porter and collaborator
suggested that for the better activity quinoxaline should have basic
nitrogen substituent; they also suggested that replacement of the
amino methyl linker by sulfonamide group increases the activity;
hence we put sulphonamido (eSO2NHe) substituents at 6th posi-
tion of quinoxaline. Further wide range of substituted phenyl ana-
logs were prepared by them, suggesting that such bulky group
Fig. 1. Reported and proposed antitumor quinoxaline derivatives.

170
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
Fig. 2. Rationale designing of the proposed compounds based upon High Throughput Screening (HTS) hit identified by Porter and collaborator.
with the core structure as shown in Fig. 3. Based on these hypoth-
eses, we designed core structure containing 6-sulphonamido qui-
noxaline as the scaffold to form the three interactions: (1) the
hydrogen bond with the hinge region (Met-1160), (2) bulky moiety
at 6th position of the quinoxalines in a fashion similar to crizotinib
which binds in the ATP-binding cleft, so that the bulky group could
be oriented deep in the back of the ATP binding site and makes
predominantly hydrophobic interactions with the protein
mimicking the 2,6-di-chloro-3-fluorophenyl group of crizotinib, (3)
hydrophobic space-filling with a quinoxaline skeleton as shown in
Fig. 3.
sulfonamide 3(aep) were confirmed by the absence of character-
istic IR absorption peak at w700 cmꢀ1 of Cl and presence of NH
stretch bands at w3300 cmꢀ1; further occurrence of broad singlet
peak at wd
5 ppm corresponding eNH group in 1H NMR substan-
tiated the formation of sulphonamido-quinoxalines 3(aep). 13C
NMR and HRMS gave information about carbon atoms and all the
Mþ ion peaks corresponding to molecular weight of confirmed
novel compounds.
3.2. Pharmacology
3.2.1. Primary single high dose (10ꢀ5 M) full NCI 60 cell panel
in vitro assay
3. Results and discussion
All the synthesized compounds 3(aep) were submitted to the
National Cancer Institute (NCI, Bethesda, MD) for the human tumor
cell screen. Among them eight compounds 3b (NSC: 763437), 3c
(NSC: 763438), 3f (NSC: 763442), 3i (NSC: 763441), 3j (NSC:
763440), 3l (NSC: 763439), 3n (NSC: 763435) and 3o (NSC: 763436)
3.1. Chemistry
The synthesis of compounds 3(aep) is given in Scheme 1. The
present study includes synthesis of sulphonyl chloride derivatives
of 2,3-diphenylquinoxaline 2 by additioneelimination reaction
mechanism. 2,3-Diphenylquinoxaline 1 was treated with chlor-
osulfonic acid under ice cold condition to give 6-sulfonyl chloride-
2,3-diphenylquinoxaline 2 [27]. It was further refluxed with the
different amines by using 10% aq. NaOH to yield sulphonyl chloride
derivatives of quinoxaline 3(aep) by nucleophilic substitution re-
action. Physical data of the synthesized compounds 3(aep) is
shown in Table 1.
The derivatives were characterized by spectral studies using IR,
1H NMR, 13C NMR and HRMS. The structures of 2,3-
diphenylquinoxaline-6-sulfonyl chloride 2 was confirmed by the
IR absorption peak at w1350, 1150 cmꢀ1 (S]O) and w700 cmꢀ1
corresponding to Cl. N-Substituted-2,3-diphenylquinoxaline-6-
were selected and tested initially at a single high dose (10
mM
concentration) in the full NCI 60 cell panel as shown in Table 2.
Effective one-dose assay has been added to the NCI 60 cell screen in
order to increase compound throughput and reduce data turn-
around time to suppliers while maintaining efficient identification
of active compounds. All the selected compounds were tested
initially at a single high dose in the full NCI 60 cell panel including
leukemia, non-small cell lung, colon, CNS, melanoma, ovarian,
renal, prostate and breast cancer cell lines. Only compounds which
satisfy pre-determined threshold inhibition criteria would progress
to the five-dose screen. The threshold inhibition criterion for pro-
gression to the five-dose screen were designed to efficiently cap-
ture compounds with anti-proliferative activity and is based on

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
171
growth inhibition curves and three response parameters (GI50, TGI
and LC50) were calculated for each cell line. The GI50 value (growth
inhibitory activity) corresponds to the concentration of the com-
pound causing 50% decrease in net cell growth, the TGI value
(cytostatic activity) is the concentration of the compound resulting
in total growth inhibition and LC50 value (cytotoxic activity) is the
concentration of the compound causing net 50% loss of initial cells
at the end of the incubation period of 48 h [28e30].
Compound 3f (NSC: 763442) exhibited high activity against
Leukemia HL-60 (GI50: 2.09
1.43 M); Non Small Cell Lung Cancer HOP-62 (GI50: 3.95
HOP-92 cell line (GI50: 2.03 M); CNS Cancer SNB-75 cell line (GI50
2.12 M); Prostate Cancer PC-3 cell line (GI50: 1.47
T-47D Cancer cell line (GI50: 1.62 M) as shown in Figs. 5 and 6.
mM) and RPMI-8226 cell lines (GI50
:
m
mM) and
m
:
m
mM) and Breast
m
Similarly compound under investigation 3l (NSC: 763439) exhibi-
ted significant anticancer activity against most of the tested cell
lines representing nine different subpanels with GI50 values be-
tween 1.11 to 4.54 mM and found to be potential candidate of the
series as shown in Figs. 7 and 8. With regard to the sensitivity
against some individual cell lines the compound 3l showed highest
activity against Leukemia RPMI-8226 cell lines (GI50: 1.11
least against Non Small Cell Lung Cancer HOP-62 cell line (GI50
4.54 M). It is to be noted that compound 3l shows significant ac-
tivity (GI50: 1.11 M) as compared to the High Throughput
Screening (HTS) hit identified by Porter and collaborator with
M [19]. Toxicity is measured in terms of lethality; both
mM) and
:
m
m
IC50 ¼ 1.3
m
compounds are not lethal and safe in nature as it is obvious by
examining the LC50 value as shown in Figs. 5 and 7.
3.2.3. Structure activity relationship
The various sulphonamido-quinoxaline derivatives 3(aep) have
been synthesized in the present study and subjected to in vitro
anticancer activity assay at National Cancer Institute. The results
were summarized in Table 2 and Figs. 5 and 7. Structure activity
correlation, in terms of NCI selected compounds (3b, 3c, 3f, 3i, 3j, 3l,
3n and 3o), based on the number of cell lines proved sensitive to-
ward each of the synthesized individual compounds, revealed that,
the quinoxaline ring is a satisfactory backbone for antitumor ac-
tivity. The presence of substituted amino moiety at the C-6 position
is necessary for the activity as hydrophobic region. The presence of
electron withdrawing group on substituted amino moiety at 6th
position enhances the anticancer activity it is obvious by comparing
anticancer results of compound 3f with 3b, 3c, 3i, 3j, 3n and 3o.
Sulphonamido (eSO2NHe) group at 6th position of quinoxaline is
acting as conformational lock and extending the bulky group into
the hydrophobic pockets of c-Met kinase, making predominantly
hydrophobic interactions with the protein mimicking the 2,6-di-
chloro-3-fluorophenyl group of crizotinib as it is obvious from
docking study. If we compare the anticancer results of five dose
Fig. 3. Proposed hypothetical model of the highly active PF-2341066 (crizotinib)/c-Met
cocrystal structure and quinoxaline 3(aep) bound to c-Met kinase.
careful analysis of historical Development Therapeutic Program
(DTP) screening data. The data are reported as a mean-graph of the
percent growth of treated cells, and presented as percentage
growth inhibition (GI%) caused by the test compounds. The ob-
tained results showed that out of eight compounds 3f (NSC:
763442) and 3l (NSC: 763439) passed successfully this primary
anticancer assay and were consequently carried over to the five-
dose screen against a panel of about 60 different tumor cell lines.
For each compound in the 5-dose screen, anticancer activity was
deduced from doseeresponse curves and expressed by three pa-
rameters (GI50, TGI, LC50) calculated for each cell line. The GI50 value
indicates the concentration of the compound required to cause 50%
inhibition of net cell growth. The TGI value represents the con-
centration of the compound resulting in total inhibition of net cell
growth. The LC50 value refers to the concentration of the compound
leading to 50% net cell death [28e30].
selected compounds 3f (GI50 1.43
mM against RPMI-8226 Leukemic
cell line) and 3l (GI50 1.11 M against RPMI-8226 Leukemic cell
m
line); it is seen that 3l is showing significant anticancer activity as
compared to 3f as shown in Figs. 5 and 7; here once again it is
proved that for better activity quinoxaline should have basic ni-
trogen substituent at 6th position.
3.3. Docking study and drug likeliness
The molecular docking tool, GLIDE (Schordinger Inc., USA) was
used for ligand docking studies into c-Met kinase receptor binding
pocket. The crystal structure of c-Met kinase was obtained from
protein data bank (PDB: 2wgj) [28]. The protein preparation was
carried out using ‘protein preparation wizard’ in Maestro 9.0 in two
steps, preparation and refinement. After ensuring chemical cor-
rectness, water molecules in the crystal structures were deleted
3.2.2. In vitro 5 dose full NCI 60 cell panel assay
All the cell lines (about 60), representing nine tumor subpanels,
were incubated at five different concentrations (0.01, 0.1, 1, 10 &
100 mM). The outcomes were used to create log concentration Vs%

172
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
Fig. 4. Sulphonamido-quinoxalines 3(aep) rationally designed by High Throughput Screening (HTS) hit identified by Porter and collaborator.
and hydrogens were added, where they were missing. Using the
OPLS 2005 force field energy of crystal structure was minimized
[29]. Grids were defined centering them on the ligand in the crystal
structure using the default box size. The ligands were built using
maestro build panel and prepared by Ligprep 2.2 module which
produce the low energy conformer of ligands using OPLS 2005 force
field. The low energy conformation of the ligands was selected and
was docked into the grid generated from protein structures using
standard precision (SP) docking mode. The final evaluation is done
with glide score (docking score) and single best pose is generated as
the output for particular ligand.
Since it was found that crizotinib mimic ATP and bind to the ATP
binding region of the kinase active site. Our compounds were
modeled by positioning them in the crizotinib binding site. From
the comparative docking study of our compounds with structurally
related lead compound such as crizotinib we could observe how
our compounds might bind to the kinase binding site, based on the
knowledge of the structure of similar active sites. We redocked
crizotinib into the active site of the enzyme and then we replaced
with our compounds in order to compare the binding mode of both
ligand and the test compound. These docking studies have revealed
that the quinoxaline ring binds to a narrow hydrophobic pocket in
the domain of c-Met kinase where N-4 of the quinoxaline ring in-
teracts with the backbone NH of Met-1160 via a hydrogen bond
(Table 1 and Fig. 9). These interactions underscore the importance
of both nitrogen atoms for binding and the subsequent inhibitory
capacity. Similarly SO2NH2 group at 6th position also shows addi-
tional two hydrogen bonding interaction (S]O with Tyr-1159 and
NH with Lys-1161). Among the docked compounds 3(aep),
compound 3p, 3f and 3l shows the highest docking score
of ꢀ8.6538, ꢀ8.2164 and ꢀ8.5808 respectively, the detail is given in
Table 1. Highest docking score of compound 3p shows that it could
be having significant anticancer activity as compared to the com-
pound 3f and 3l but it is not selected at NCI-USA for the anticancer
screening. The results of this virtual screening could support the
postulation that our active compounds may act on the same
enzyme target c-Met kinase. It is also seen that bulky moiety at 6th
position of the quinoxaline in a fashion similar to crizotinib ori-
ented deep in the back of the ATP binding site and makes pre-
dominantly hydrophobic interactions with the protein mimicking
the 2,6-di-chloro-3-fluorophenyl group of crizotinib.
Fig. 9 demonstrate binding mode of crizotinib and quinoxaline
derivatives 3f and 3l. Crizotinib forms hydrogen bonding with Met-
1160 via N-1 of pyridine ring and similar hydrogen bonding inter-
action is also shown by N-4 of quinoxaline (3f and 3l) with Met-
ꢀ
1160. Residues within 5 A areas of crizotinib and quinoxaline de-
rivatives 3f and 3l are shown in Fig. 9. Some common residues
ꢀ
involved in this type of interaction within 5 A area are VAL-1092,
TYR-1159, ALA-1108, MET-1211, ARG-1208, ILE-1084, and ASP-1231.
We further analyzed physically significant descriptors and
pharmaceutically relevant properties of all synthesized com-
pounds, among which were molecular weight, LogP, H-bond
donors, H-bond acceptors according to Lipinski’s rule of five
(Table 3). Lipinski’s rule of five is a rule of thumb to evaluate
drug likeness, or determine if a chemical compound with a
certain pharmacological or biological activity has properties that
would make it a likely orally active drug in humans. The rule
describes delicate balance among the molecular properties of a

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
173
Scheme 1. Reaction scheme for the synthesis of target compounds 3(aep).
compound that directly influence its pharmacodynamics and
pharmacokinetics and ultimately affect their absorption, distri-
bution, metabolism, and excretion in human body like a drug
[30]. In general, these parameters allow to ascertain a poor oral
absorption, or membrane permeability, that occurs when the
evaluated molecules present values higher than five H-bond
donors (HBD), 10 H-bond acceptors (HBA), molecular weight
(MW) > 500 Da and LogP (cLogP) > 5 (Lipinski’s ‘rule-of-five’)
[31]. The compounds were evaluated for their drug-like behavior
through analysis of pharmacokinetic parameters required for
absorption, distribution, metabolism and excretion (ADME) by
use of QikProp [32].
For the 16 compounds, the partition co-efficient (QPlogPo/w)
and water solubility (QPlogS), critical for estimation of absorption
and distribution of drugs within the body ranged between 2.591 to
5.39 and ꢀ6.788 to ꢀ4.897. Cell permeability (QPPCaco), a key
factor governing drug metabolism and its access to biological
membranes, ranged from 40.588 to 981.419, QPPMDCK ranges from
19.782 to 1085.791. Overall, the percentage human oral absorption
for the compounds ranged from 81.389 to 100%. All these phar-
macokinetic parameters are within the acceptable range defined
for human use (see Table 3 footnote), thereby indicating their po-
tential as drug-like molecules.
concentration at the NCI over 60 cell line panel, and compounds 3f
and 3l were subsequently tested in 5-dose testing mode. With re-
gard to the sensitivity against some individual cell lines the com-
pound 3l showed highest activity against Leukemia RPMI-8226 cell
lines (GI50: 1.11
be noted that compound 3l shows significant activity (GI50
1.11 M) as compared to the High Throughput Screening (HTS) hit
identified by Porter and collaborator with IC50 ¼ 1.3 M. Molecular
mM) as compared to other tested compounds. It is to
:
m
m
docking studies further supports our assumption that the synthe-
sized compounds have analogous binding mode to the c-Met kinase
inhibitors and demonstrates the various interactions between the
ligands and enzyme active sites and thereby help to design novel
potent inhibitors. The overall outcome of this model revealed that:
(i) the quinoxaline ring is a satisfactory backbone for antitumor
activity; (ii) the presence of substituted amino moiety at the C-6
position is necessary for the activity as hydrophobic region; (iii) the
presence of electron withdrawing group on substituted amino
moiety at 6th position enhances the anticancer activity; (iv) sul-
phonamido (eSO2NHe) group at 6th position of quinoxaline is
acting as conformational lock and extending the bulky group into
the hydrophobic pockets of c-Met kinase, making predominantly
hydrophobic interactions with the protein mimicking the 2,6-di-
chloro-3-fluorophenyl group of crizotinib. Lipinski’s rule and in
silico ADME pharmacokinetic parameters are within the acceptable
range defined for human use thereby indicating their potential as
drug-like molecules. These encouraging results of biological
screening of the tested compounds could offer an excellent
framework in this field that may lead to discovery of potent anti-
tumor agent. Finally it is conceivable that further derivatization of
such compounds will be of interest with the hope to get more se-
lective anticancer agents.
4. Conclusion
In conclusion a new series of sulphonamido-quinoxalines 3(ae
p) were synthesized. Among all of these derivatives, compounds 3b
(NSC: 763437), 3c (NSC: 763438), 3f (NSC: 763442), 3i (NSC:
763441), 3j (NSC: 763440), 3l (NSC: 763439), 3n (NSC: 763435) and
3o (NSC: 763436) were tested at a single dose of 10ꢀ5
M

174
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
Table 1
Physicochemical properties of the synthesized compounds 3(aep) and glide docking results based on glide dock score, glide energy, glide pose and hydrogen bonding
interaction.
S. no.
Compounds
Molecular
formula
Melting
point (ꢁC)
Percentage
yield (%)
Docking
score
Glide energy
(kcal/mol)
Glide
pose
H-bond interaction
3a
C22H17N3O3S
146e150
72
ꢀ5.7239
ꢀ43.3463
220
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H
atom of amino acid Tyr-1159
NH of sulphonamido with
carbonyl oxygen of Lys-1161
3b
C27H19N3O3S
172e176
68
ꢀ4.9109
ꢀ42.7556
284
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3c
C30H21N3O2S
182e186
53
ꢀ7.9830
ꢀ48.4402
393
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H
atom of amino acid Tyr-1159
NH of sulphonamido with
carbonyl oxygen of Lys-1161
3d
3e
3f
C30H21N3O2S
C26H19N3O3S
C26H18ClN3O2S
190e194
202e204
222e226
58
56
74
ꢀ5.7691
ꢀ7.0208
ꢀ8.5808
ꢀ57.1301
ꢀ54.8931
ꢀ50.0947
176
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H
atom of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
45
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H
atom of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
295
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H
atom of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
175
Table 1 (continued )
S. no.
Compounds
Molecular
formula
Melting
point (ꢁC)
Percentage
yield (%)
Docking
score
Glide energy
(kcal/mol)
Glide
pose
H-bond interaction
3g
C26H18N4O4S
178e182
79
ꢀ7.4131
ꢀ58.8267
384
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3h
C26H18N4O4S
192e196
69
ꢀ7.3535
ꢀ58.8077
56
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3i
C27H21N3O3S
C26H20N4O2S
C27H19N3O4S
202e204
208e212
234e238
59
68
71
ꢀ6.4248
ꢀ6.1365
ꢀ6.0096
ꢀ50.2353
ꢀ49.0306
ꢀ47.4862
94
242
150
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3j
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3k
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3l
C25H18N4O2S
246e250
74
ꢀ8.2164
ꢀ50.1261
232
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3m
C26H20N4O2S
228e232
62
ꢀ7.1890
ꢀ53.5692
168
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
(continued on next page)

176
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
Table 1 (continued )
S. no.
Compounds
Molecular
formula
Melting
point (ꢁC)
Percentage
yield (%)
Docking
score
Glide energy
(kcal/mol)
Glide
pose
H-bond interaction
3n
C21H17N5O2S2
146e148
66
ꢀ5.7918
ꢀ48.3404
05
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H
atom of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
3o
3p
C22H17N3O3S
162e164
68
62
ꢀ5.2312
ꢀ8.8983
ꢀ43.2311
ꢀ58.6781
193
376
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H atom
of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
C28H21N3O3S
188e192
N- of quinoxaline and H atom of
amino acid backbone of MET-1160
S]O of sulphonamido with H
atom of amino acid Tyr-1159
NH of sulphonamido with carbonyl
oxygen of Lys-1161
5. Experimental protocols
poured into the crushed ice and stirred until product solidifies; it
was then filtered, washed with dilute NaOH solution and recrys-
tallized from ethanol.
All chemicals and solvents were supplied by Merck, S.D. Fine
Chemical Limited, Mumbai. All the solvents were distilled and dried
before use. The reactions were monitored with the help of thin-
layer chromatography using pre-coated aluminum sheets with
GF254 silica gel, 0.2 mm layer thickness (E. Merck). The solvents
used throughout the experiment for running TLC were ethyl acetate
and petroleum ether in the ratio of 3:2, chloroform and methanol in
the ratio of 9.5:0.5 and 9:1 as developing solvents. UV Cabinet was
used for the visualization of TLC spots. Melting points of the syn-
thesized compounds were recorded on the Veego (VMP-MP)
melting point apparatus. IR spectrum was acquired on a Shimadzu
Infra Red Spectrometer, (model FTIR-8400S). Both 1H NMR (DMSO)
and 13C NMR (DMSO) spectra of the synthesized compounds were
performed with Bruker Avance-II 400 NMR Spectrometer operating
at 400 MHz in SAIF, Punjab University (Chandigarh). Chemical shifts
5.2.1. N-(2,3-Diphenylquinoxalin-6-ylsulfonyl) acetamide (3a)
IR (KBr) nmax 3345.36 (NH stretch), 3056.44 (CH Arom.), 2934.48
(CH Aliph.), 1660.64 (C]O), 1582.34 (NH bend), 1347.66, 1174.97
(S]O) cmꢀ1; 1H NMR (DMSO-d6)
d
ppm: 10.21 (br s, 1H, NH), 7.32e
8.39 (m, 13H, AreH), 2.48 (s, 3H, CH3); 13C NMR (DMSO-d6)
d
ppm:
169.56, 156.23, 146.78, 145.24,143.63, 139.24,130.62,129.24,128.21,
127.24, 126.12, 125.24, 21.26; HRMS (EI) m/z calcd for C22H17N3O3S:
403.0991; found: 403.0995.
5.2.2. N-(2,3-Diphenylquinoxalin-6-ylsulfonyl) benzamide (3b)
IR (KBr) nmax 3413.91 (NH stretch), 3046.83 (CH stretch), 1672.74
(C]O), 1592.63 (NH bend), 1350.19, 1141.94 (S]O) cmꢀ1 1H NMR
;
(DMSO-d6)
d
ppm: 10.34 (br s, 1H, NH), 7.21e8.38 (m, 18H, AreH);
ppm: 169. 78, 158.47, 148.74, 145.46, 144.84,
were measured relative to internal standard TMS (
d
: 0). Chemical
13C NMR (DMSO-d6)
d
shifts are reported in scale (ppm). Mass spectra of the synthesized
compounds were recorded at MAT 120 in SAIF, Punjab University.
d
141.93, 136.53, 133.74, 130.35, 129.75, 128.84, 128.33, 127.83, 127.22,
126.74, 125.23; HRMS (EI) m/z calcd for C27H19N3O3S: 465.1147;
found: 465.1151.
5.1. Synthesis of 2,3-diphenylquinoxaline-6-sulfonyl chloride (2)
5.2.3. N-(Naphthalen-1-yl)-2,3-diphenylquinoxaline-6-
sulfonamide (3c)
It is prepared as per the procedure mentioned by Ganapaty et al.
[27].
IR (KBr) nmax 3343.00 (NH stretch), 3055.85 (CH stretch),
1681.56 (C]O), 1598.56 (NH bend), 1345.14, 1164.91 (S]O) cmꢀ1
;
5.2. General procedure for the synthesis of N-substituted-2,3-
diphenylquinoxaline-6-sulfonamide 3(aep)
1H NMR (DMSO-d6)
d
ppm: 7.22e8.16 (m, 20H, AreH), 5.31 (br s,
ppm: 158.78, 147.34, 145.24,
1H, NH); 13C NMR (DMSO-d6)
d
141.54, 140.20, 139.45, 135.32, 130.64, 129.75, 128.23, 128.11,
127.08, 127.03, 126.65, 126.25, 126.02, 125.74, 124.23, 122.01,
118.64, 109.44; HRMS (EI) m/z calcd for C30H21N3O2S: 487.1354;
found: 487.1358.
A primary amino containing moiety (0.01 mol) was refluxed
with 2,3-diphenylquinoxaline-6-sulfonylchloride 2 (0.01 mol) in
50 mL of 10% aq. NaOH solution for 5 h. The reaction mixture was

Table 2
Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10ꢀ5
m
M (Single Dose Assay).
Compound
Cancer Cell Line
Leukemia
41.34
49.43
45.75
44.86
58.54
71.12
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
58.32
70.98
49.56
47.78
44.34
81.12
61.94
66.73
55.63
60.82
86.04
88.12
49.84
60.73
45.52
56.93
65.72
81.92
32.64
39.92
34.92
35.52
47.78
53.78
70.73
80.72
64.42
86.83
89.93
-3.25
35.83
40.63
34.82
28.12
46.37
49.98
24.82
35.26
26.83
20.54
39.62
40.95
Non-Small Cell Lung
Cancer
A549/ATCC
EKVX
HOP-62
21.12
26.54
2.87
15.34
71.23
2.24
43.97
21.34
22.23
23.56
47.82
36.45
48.82
2.12
69.93
23.03
23.23
45.54
28.82
17.57
30.57
2.52
59.84
16.56
10.77
26.63
18.67
41.93
57.83
12.83
-2.45
26.64
32.82
47.12
50.83
54.67
80.82
22.76
-1.38
46.52
59.42
63.42
65.63
34.72
23.72
38.73
52.93
11.62
2.92
7.74
8.53
2.84
53.64
11.67
17.83
8.63
61.97
19.56
20.87
31.97
23.54
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
15.63
13.74
3.73
19.54
15.32
36.67
44.94
48.43
20.98
17.43
27.87
4.56
28.94
19.92
55.72
44.72
63.92
8.62
12.67
1.83
36.56
32.53
39.61
6.93
4.83
8.92
4.64
6.23
26.64
37.92
59.62
44.72
70.43
38.74
30.32
54.23
60.63
72.12
62.43
81.72
61.52
47.92
49.87
34.34
82.45
29.92
26.78
17.83
29.92
30.82
15.92
3.72
14.53
26.21
21.46
16.75
3.84
HT 29
KM 12
SW-620
21.54
13.56
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
6.89
20.98
3.89
21.98
34.67
21.56
2.78
3.88
5.57
9.78
39.88
7.23
23.82
15.92
24.72
41.92
31.93
ND
1.63
14.52
7.76
16.52
29.64
15.62
2.92
8.92
21.72
14.82
25.63
26.82
1.86
5.83
3.29
2.12
26.85
10.32
14.12
40.73
25.93
31.93
45.12
39.45
27.63
76.83
61.92
43.63
61.82
56.53
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
20.54
7.67
2.45
12.89
15.84
7.63
48.93
15.24
19.65
32.87
9.98
2.23
20.45
14.87
5.98
48.56
21.66
28.86
23.34
1.87
15.34
2.94
4.84
7.72
2.94
4.26
42.28
1.94
30.23
11.23
2.94
4.23
11.13
7.94
6.93
39.13
8.83
24.34
12.23
2.85
2.85
4.73
8.53
1.73
28.93
2.23
20.72
38.42
6.64
63.32
32.85
62.84
54.24
39.29
30.29
-6.25
42.45
41.13
25.98
22.53
24.97
12.67
61.42
18.87
23.42
30.96
35.63
23.93
14.64
73.95
17.85
30.56
Ovarian Cancer
IGROV1
16.54
19.25
16.53
8.43
1.78
30.88
31.23
2.98
20.56
22.62
5.86
11.78
13.46
29.98
12.73
24.12
40.98
18.63
1.84
2.86
17.25
5.94
4.27
13.26
2.37
8.83
1.84
16.54
4.85
5.83
11.88
5.98
14.84
1.94
13.43
2.84
3.93
8.63
4.85
20.83
28.84
30.28
18.28
34.84
55.21
18.48
31.82
48.92
40.12
24.34
52.75
73.20
44.93
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
8.02
27.53
12.83

178
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
5.2.4. N-(Naphthalen-2-yl)-2,3-diphenylquinoxaline-6-
sulfonamide (3d)
IR (KBr) nmax 3422.14 (NH stretch), 3056.08 (CH stretch), 1673.78
(C]O), 1601.68 (NH bend), 1362.72, 1162.25 (S]O) cmꢀ1 1H NMR
;
(DMSO-d6)
NMR (DMSO-d6)
d
ppm: 7.32e8.14 (m, 20H, AreH), 5.38 (br s,1H, NH); 13
ppm: 158.46, 148.34, 145.24, 143.34, 142.25,
C
d
140.66, 135.78, 130.63, 129.18, 129.08, 128.35, 127.74, 126.78, 126.58,
126.46, 126.02, 125.48, 124.38, 122.44, 119.14, 108.14; HRMS (EI) m/z
calcd for C30H21N3O2S: 487.1354; found: 487.1359.
5.2.5. N-(4-Hydroxyphenyl)-2,3-diphenylquinoxaline-6-
sulfonamide (3e)
IR (KBr) nmax 3564.48 (OH stretch), 3423.24 (NH stretch),
3050.81 (CH stretch), 1668.24 (C]O), 1578.24 (NH bend), 1332.23,
1168.57 (S]O) cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 7.12e8.46 (m,
17H, AreH), 5.68 (s, 1H, OH), 4.56 (br s, 1H, OH); 13C NMR (DMSO-
d6) ppm: 158.57, 149.34, 148.43, 145.63, 142.84, 138.64, 134.95,
d
130.63, 129.63, 128.54, 127.21, 126.64, 126.22, 125.42, 119.22; HRMS
(EI) m/z calcd for C26H19N3O3S: 453.1147; found: 453.1151.
5.2.6. N-(4-Chlorophenyl)-2,3-diphenylquinoxaline-6-sulfonamide
(3f)
IR (KBr) nmax 3429.83 (NH stretch), 3028.34 (CH stretch),1672.58
(C]O), 1588.65 (NH bend), 1350.67, 1180.24 (S]O), 763.42 (Ce
Cl) cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 7.04e8.10 (m, 17H, AreH),
5.26 (br s, 1H, NH); 13C NMR (DMSO-d6)
d
ppm: 158.64, 148.42,
144.73,142.63,140.56,138.34, 134.78,133.23, 132.67, 130.24,129.78,
128.56, 127.34, 126.22, 122.56; HRMS (EI) m/z calcd for
C26H18ClN3O2S: 471.0808; found: 471.0804.
5.2.7. N-(4-Nitrophenyl)-2,3-diphenylquinoxaline-6-sulfonamide
(3g)
IR (KBr) nmax 3472.91 (NH stretch), 3048.89 (CH stretch),1675.87
(C]O),1568.28 (NH bend),1549.23,1348.83 (NO2),1337.76,1180.26
(S]O) cmꢀ1; 1H NMR (DMSO-d6)
d
ppm: 7.34e8.25 (m, 17H, AreH),
5.56 (br s, 1H, NH); 13C NMR (DMSO-d6)
d
ppm: 159.68, 149.24,
146.45,144.24,142.78,140.34,138.24,130.78,129.45,128.28,127.84,
126.45, 125.68, 124.46, 119.34; HRMS (EI) m/z calcd for
C26H18N4O4S: 482.1049; found: 482.1053.
5.2.8. N-(3-Nitrophenyl)-2,3-diphenylquinoxaline-6-sulfonamide
(3h)
IR (KBr) nmax 3414.12 (NH stretch), 3050.82 (CH stretch), 1678.34
(C]O),1592.78 (NH bend),1546.23,1354.68 (NO2),1338.78,1174.86
(S]O) cmꢀ1; 1H NMR (DMSO-d6)
d
ppm: 7.37e8.13 (m, 17H, AreH),
5.48 (br s, 1H, NH); 13C NMR (DMSO-d6)
d
ppm: 158.56, 150.23,
148.68, 147.38, 145.24, 141.73, 139.28, 134.48, 132.22, 130.64, 129.62,
128.84, 127.53, 126.22, 125.78, 116.34, 115.68; HRMS (EI) m/z calcd
for C26H18N4O4S: 482.1049; found: 482.1045.
5.2.9. N-(2-Methoxyphenyl)-2,3-diphenylquinoxaline-6-
sulfonamide (3i)
IR (KBr) nmax 3404.34 (NH stretch), 3050.22 (CH stretch),
1684.82 (C]O), 1599.53 (NH bend), 1342.68, 1179.78 (S]O) cmꢀ1
;
1H NMR (DMSO-d6)
NH), 3.81 (s, 3H, OCH3); 13C NMR (DMSO-d6)
d
ppm: 7.35e8.19 (m, 17H, AreH), 5.34 (br s, 1H,
ppm: 159.88, 151.34,
d
148.87, 145.46, 144.78,140.34, 135.24,132.88, 130.66,129.47, 128.84,
127.62, 126.56, 126.22, 124.66, 122.34, 118.24, 55.88; HRMS (EI) m/z
calcd for C27H21N3O3S: 467.1304; found: 467.1309.
5.2.10. N0-2,3-Triphenylquinoxaline-6-sulfonohydrazide (3j)
IR (KBr) nmax 3411.83 (NH stretch), 3049.82 (CH stretch), 1662.43
(C]O), 1596.82 (NH bend), 1338.60, 1147.65 (S]O) cmꢀ1; 1H NMR
(DMSO-d6)
d
ppm: 7.33e8.21 (m, 18H, AreH), 6.45 (s, 1H, NH), 5.12
ppm: 158.68, 154.38, 149.44,
(s, 1H, SONH); 13C NMR (DMSO-d6)
d

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
179
Fig. 5. Five dose assay of compound 3f (NSC: 763442).

180
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
Fig. 6. Five dose assay graph of compound 3f (NSC: 763442) against nine panel cancer cell line at NCI.
145.77, 142.73, 140.98, 132.38, 131.34, 130.48, 129.24, 128.52, 126.63,
125.28, 122.18, 113.24; HRMS (EI) m/z calcd for C26H20N4O2S:
452.1307; found: 452.1312.
5.2.13. N-(4-Methylpyridin-2-yl)-2,3-diphenylquinoxaline-6-
sulfonamide (3m)
IR (KBr) nmax 3408.24 (NH stretch), 3018.78 (CH Arom.), 2918.34
(CH Alip.),1663.68 (C]O), 1581.64 (NH bend), 1352.78, 1152.63 (S]
5.2.11. 2-(2,3-Diphenylquinoxaline-6-sulfonamido)benzoic acid
(3k)
O) cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 7.35e8.25 (m, 16H, AreH),
5.31 (br s, 1H, NH), 2.11 (s, 3H, CH3); 13C NMR (DMSO-d6)
d
ppm:
IR (KBr) nmax 3385.33 (OH acid), 3327.28 (NH stretch), 3055.28
(CH stretch), 1645.83 (C]O), 1578.82 (NH bend), 1346.62, 1146.63
158.48, 154.48, 152.68, 149.38, 146.58, 145.48, 144.58, 140.38,
135.58, 132.47, 130.23, 129.57, 128.56, 127.23, 122.12, 118.57, 22.45;
HRMS (EI) m/z calcd for C26H20N4O2S: 452.1307; found: 452.1303.
(S]O) cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 10.21 (s, 1H, OH), 7.25e
8.21 (m, 17H, AreH), 5.34 (s, 1H, NH2); 13C NMR (DMSO-d6)
d
ppm:
170.68, 156.88, 149.34, 146.48, 145.18, 143.57, 140.32, 138.47,
135.14, 132.86, 131.28, 129.78, 128.57, 127.36, 126.24, 119.24, 116.21,
112.28; HRMS (EI) m/z calcd for C27H19N3O4S: 481.1096; found:
481.1091.
5.2.14. 2-(2,3-Diphenylquinoxalin-6-ylsulfonyl)
hydrazinecarbothioamide (3n)
IR (KBr) nmax 3398.9 (NH stretch), 3034.24 (CH stretch), 1676.24
(C]O), 1572.88 (NH bend), 1358.24, 1162.34 (S]O) cmꢀ1; 1H NMR
(DMSO-d6)
d
ppm: 7.15e8.16 (m, 13H, AreH), 10.12 (s, 2H, NH2), 5.12
ppm: 179.56,
5.2.12. 2,3-Diphenyl-N-(pyridin-2-yl)quinoxaline-6-sulfonamide
(3l)
(s, 1H, NH), 4.42 (s, 1H, NH); 13C NMR (DMSO-d6)
d
158.48, 148.85, 144.24, 142.68, 140.49, 134.23, 132.56, 130.29,
129.58, 128.45, 126.56; HRMS (EI) m/z calcd for C21H17N5O2S2:
435.0824; found: 435.0829.
IR (KBr) nmax 3409.28 (NH stretch), 3056.26 (CH stretch),
1658.84 (C]O), 1588.82 (NH bend), 1356.34, 1148.65 (S]O) cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 7.30e8.25 (m, 17H, AreH), 5.49 (br s,
ppm: 158.78, 155.24, 150.78, 148.36,
1H, NH); 13C NMR (DMSO-d6)
d
5.2.15. N-(2,3-Diphenylquinoxalin-6-ylsulfonyl)ethanethioamide
(3o)
IR (KBr) nmax 3424.26 (NH stretch), 3035.56 (CH Arom.),
2934.56 (CH Alip.), 1668.24 (C]O), 1596.22 (NH bend), 1356.34,
145.44,143.44,140.23,139.93,132.24,131.68, 129.48,128.34,127.28,
126.38, 118.28, 109.19; HRMS (EI) m/z calcd for C25H18N4O2S:
438.1150; found: 438.1155.

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
181
Fig. 7. Five dose assay of compound 3l (NSC: 763439).

182
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
Fig. 8. Five dose assay graph of compound 3l (NSC: 763439) against nine panel cancer cell line at NCI.
1162.46 (S]O) cmꢀ1
;
1H NMR (DMSO-d6)
d
ppm: 7.32e8.16 (m,
the COMPARE program. Compounds which exhibit significant
growth inhibition are evaluated against the 60 cell panel at five
concentration levels. The human tumor cell lines of the cancer
screening panel are grown in RPMI 1640 medium containing 5%
13H, AreH), 5.38 (br s, 1H, NH), 2.68 (s, 3H, CH3); 13C NMR
(DMSO-d6)
d ppm: 196.65, 159.34, 149.58, 145.24, 143.56,
140.68, 132.26, 130.88, 129.44, 128.34, 126.61, 125.24, 35.34;
HRMS (EI) m/z calcd for
419.0766.
C
22H17N3O2S2: 419.0762; found:
fetal bovine serum and 2 mM
experiment, cells are inoculated into 96 well microtiter plates in
100 L at plating densities ranging from 5000 to 40,000 cells/well
L-glutamine. For a typical screening
m
5.2.16. N-(3-Acetylphenyl)-2,3-diphenylquinoxaline-6-sulfonamide
(3p)
depending on the doubling time of individual cell lines. After cell
inoculation, the microtiter plates are incubated at 37 ꢁC, 5% CO2,
95% air and 100% relative humidity for 24 h prior to addition of
experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line
at the time of drug addition (Tz). Experimental drugs are solubi-
lized in dimethyl sulfoxide at 400 fold the desired final maximum
test concentration and stored frozen prior to use. At the time of
drug addition, an aliquot of frozen concentrate is thawed and
diluted to twice the desired final maximum test concentration with
IR (KBr) nmax 3444.24 (NH stretch), 3055.24 (CH Arom.), 2901.56
(CH Alip.), 1672.88 (C]O), 1578.24 (NH bend), 1362.22, 1142.78 (S]
O) cmꢀ1; 1H NMR (DMSO-d6)
d
ppm: 7.01e8.34 (m,17H, AreH), 5.41
(br s, 1H, NH), 2.58 (s, 3H, CH3); 13C NMR (DMSO-d6)
d
ppm: 194.68,
158.34, 148.24, 144.34, 142.56, 140.68, 138.34, 136.89, 135.34,
132.34, 130.68, 128.46, 127.34, 126.24, 126.02, 122.28, 119.48, 116.48,
24.24; HRMS (EI) m/z calcd for C28H21N3O3S: 479.1304; found:
479.1309.
5.3. Protocol of in vitro anticancer screening at NCI
complete medium containing 50
10-fold or ½ log serial dilutions are made to provide a total of five
drug concentrations plus control. Aliquots of 100 l of these
different drug dilutions are added to the appropriate microtiter
wells already containing 100 l of medium, resulting in the
required final drug concentrations.
mg/ml gentamicin. Additional four,
The in vitro anticancer screening at NCI is a two-stage process,
beginning with the evaluation of all compounds against the 60 cell
m
lines at a single dose of 10
mM. The output from the single dose
m
screen is reported as a mean graph and is available for analysis by

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
183
Fig. 9. Binding mode of crizotinib (c-Met Kinsae Inhibitor) H-bond interaction with MET-1160 shown in (A) and compound 3f (NSC: 763442), (B) 3l (NSC: 763439), (C) 3p, (D)
showing hydrogen bond interaction with N- of quinoxaline and H atom of amino acid backbone of MET-1160; S]O of sulphonamido with H atom of amino acid Tyr-1159; NH of
sulphonamido with carbonyl oxygen of Lys-1161 of c-Met Kinsae receptor (PDB: 2wgj) [H-bonding interaction is shown in pink dotted line]. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Table 3
Lipinski’s rule of five for drug likeliness and in silico ADME properties of 3(aep) by QikProp (Schordinger 9.0).
Criteria
S. no.
Lipinski’s rule of five (Drug Likeliness)
In silico ADME by QikProp, Schordinger 9.0
Compounds
Molecular
weight
QPlogP
O/Wa
H-Bond
donor
H-Bond
acceptor
QPlogSb
QPlogHERGc
QPPCacod
QPMDCKe
% Human
oral
absorptionf
3a
3b
3c
403.455
465.525
487.575
3.527
4.772
5.391
01
01
01
7
ꢀ5.647
ꢀ6.989
427.026
200.927
94.677
7
ꢀ6.788
ꢀ6.685
ꢀ8.075
ꢀ7.708
486.878
969.418
227.464
479.587
100
6.5
100
3d
487.575
5.504
01
6.5
ꢀ6.789
ꢀ7.729
981.419
494.532
100
(continued on next page)

184
R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
Table 3 (continued )
Criteria
S. no.
Lipinski’s rule of five (Drug Likeliness)
In silico ADME by QikProp, Schordinger 9.0
Compounds
Molecular
weight
QPlogP
O/Wa
H-Bond
donor
H-Bond
acceptor
QPlogSb
ꢀ5.305
ꢀ6.199
QPlogHERGc
QPPCacod
322.424
954.38
QPMDCKe
% Human
oral
absorptionf
3e
3f
453.514
471.96
3.765
4.957
02
01
7.25
6.5
ꢀ6.97
147.55
93.886
ꢀ6.961
1085.791
100
3g
482.513
482.513
3.869
3.799
01
01
7.5
7.5
ꢀ5.716
ꢀ5.848
ꢀ7.075
ꢀ7.203
133.383
108.407
56.572
44.809
87.633
85.613
3h
3i
467.541
452.53
5.03
01
02
01
7.25
ꢀ6.358
ꢀ6.248
ꢀ6.466
ꢀ7.999
ꢀ8.018
ꢀ5.981
241.673
458.515
40.588
635.372
318.385
19.782
100
3j
3k
4.426
4.756
7.5
100
481.525
7.5
83.58
3l
438.503
452.53
4.03
01
01
03
01
6.5
7.5
09
ꢀ4.897
ꢀ5.628
ꢀ5.654
ꢀ5.657
ꢀ6.968
ꢀ6.959
ꢀ7.3
460.727
477.758
156.438
428.521
267.902
273.417
171.476
201.632
100
3m
3n
3ꢁ
4.154
2.591
3.531
100
435.518
403.455
81.389
94.726
07
ꢀ7.001

R. Ingle et al. / European Journal of Medicinal Chemistry 65 (2013) 168e186
185
Table 3 (continued )
Criteria
S. no.
Lipinski’s rule of five (Drug Likeliness)
In silico ADME by QikProp, Schordinger 9.0
Compounds
Molecular
weight
QPlogP
O/Wa
H-Bond
donor
H-Bond
acceptor
QPlogSb
QPlogHERGc
QPPCacod
QPMDCKe
% Human
oral
absorptionf
3p
479.552
4.068
01
8.5
ꢀ5.653
ꢀ7.144
377.624
174.263
96.887
a
Predicted octanol/water partition co-efficient LogP (acceptable range: ꢀ2.0e6.5).
Predicted aqueous solubility; S in mol/L (acceptable range: ꢀ6.5e0.5).
Predicted IC50 value for blockage of HERG Kþ channels (concern below ꢀ5.0).
b
c
d
e
f
Predicted Caco-2 cell permeability in nm/s (acceptable range: <25 is poor and >500 is great).
Predicted apparent MDCK cell permeability in nm/s.
Percentage of human oral absorption (<25% is poor and >80% is high).
Following the drug addition, the plates are incubated for an
Acknowledgement
additional 48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative humidity.
For adherent cells, the assay is terminated by the addition of cold
Authors are thankful to National Cancer Institute (NCI, USA) for
in vitro anticancer activity.
TCA. Cells are fixed in situ by the gentle addition of 50 ml of cold 50%
(w/v) TCA (final concentration, 10% TCA) and incubated for 60 min
at 4 ꢁC. The supernatant is discarded, and the plates are washed five
times with tap water and air-dried. Sulforhodamine B (SRB) solu-
Appendix A. Supplementary data
tion (100 ml) at 0.4% (w/v) in 1% acetic acid is added to each well,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.04.028.
and plates are incubated for 10 min at room temperature. After
staining, unbound dye is removed by washing five times with 1%
acetic acid and the plates are air-dried. Bound stain is subsequently
solubilized with 10 mM trizma base, and the absorbance is read on
an automated plate reader at a wavelength of 515 nm. For sus-
pension cells, the methodology is the same except that the assay is
terminated by fixing settled cells at the bottom of the wells by
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