1-[(2E)-3-Phenylprop-2-enoyl]-1H-benzimidazoles as anticancer agents: synthesis, crystal structure analysis and binding studies of the most potent anticancer molecule with serum albumin

Article abstract of DOI:10.1039/c5md00293a

A series of 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazoles have been synthesized and their ability to inhibit the growth of cancer cell lines has been investigated against an NCI 60 cell panel. The compound 1-[(2E)-3-phenylprop-2-enoyl]-2-(4-chlorophenyl

Full text of DOI:10.1039/c5md00293a

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1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazoles as anticancer
agents: Synthesis, crystal structure analysis and binding studies of the
most potent anticancer molecule with serum albumin
Veerendra Kumar A. Kalalbandi, J. Seetharamappa*
Department of Chemistry, Karnatak University, Dharwadꢀ580 003, India
*Corresponding author: Eꢀmail:jseetharam@yahoo.com
Phone No.: +91ꢀ836ꢀ 2215286
Fax
: +91ꢀ836ꢀ2747884
Abstract
A series of 1ꢀ[(2E)ꢀ3ꢀphenylpropꢀ2ꢀenoyl]ꢀ1Hꢀbenzimidazoles have been
synthesized and their ability to inhibit the growth of cancer cell lines has been
investigated against NCI 60 cell panel. The compound 1ꢀ[(2E)ꢀ3ꢀphenylpropꢀ2ꢀ
enoyl]ꢀ2ꢀ(4ꢀchlorophenyl)ꢀ1Hꢀbenzimidazole (Compound 3f, NSC: 773404/1) was
found to display good antitumor activity against the nine tumor subpanels with the
selectivity ratios ranging from 0.79 to 1.53 and from 0.47 to 1.69 at GI50 and TGI
levels, respectively. Structural parameters of the potent anticancer active compound,
3f have been studied from single crystal XRD data. Further, in vitro interaction of 3f
with transport protein, human serum albumin (HSA) was investigated by
spectroscopic techniques. Static quenching mechanism was noticed in the binding of
3f to HSA. Thermodynamic parameters revealed that the hydrogen bond and van der
Waals forces played a major role in the interaction process.
Keywords: Anticancer activity; 1Hꢀbenzimidazoles; crystal structure; HSA; binding
studies.

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Introduction
In recent years, cancer is recognized as the leading disease that has posed a threat
to human life. The world health organization has reported that the global cancer
burden was doubled during the last 30 years of the 20th century.1 The development of
resistance against the prevailing anticancer drugs has generated more complication in
the treatment of cancer2,3 that keeps research window open in search of newer
anticancer agents. However, the window passage has become narrower because it is
rather more difficult to search a molecule that can selectively inhibit the proliferation
of abnormal cells only with least or no effect on normal cells. Hence, investigation of
a new chemical entity for the treatment of cancer assumes importance. Among several
bioactive compounds, benzimidazoles still remain as potential bioactive molecules
and construction of the benzimidazole pharmacophore may give a fruitful result in the
investigation of cancer related drugs.4,5
Benzimidazole compounds have occupied a remarkable place among various
heterocycles by virtue of their greater involvement in medicinal chemistry and drug
discovery. Benzimidazoles are the key building block of numerous compounds that
play a vital role in the functioning of bioactive molecules.6 Different biological
activities have been reported as these are structural isosteres of naturally occurring
nucleotides that interact easily with biopolymers of the living system.7 Substituted
derivatives of benzimidazole exhibited noteworthy anticancer activities8ꢀ10 besides a
wide range of biological activities including antiviral,11,12 antimicrobial,13
anthelmintic,14 antiꢀinflammatory15 and antihistaminic16 properties.

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In addition to benzimidazoles, compounds containing cinnamoyl group are known
to exhibit potent anticancer activity. Cinnamoyl group belongs to the class of auxin,
which is recognized as the plant hormone regulating cell growth and differentiation.17
The cinnamoyl functionality is also present in a variety of secondary metabolites of
phenylpropenoid biosynthetic origin. The presence of α, βꢀunsaturated carbonyl
moiety in cinnamoyl group would be considered as a Michael acceptor, which is an
active group often employed in the design of efficient anticancer agents.18 Further, the
literature survey revealed that the compounds containing cinnamoyl group and
cinnamoyl anthranilates exhibited significant anticancer activity.19 In view of
biological importance of cinnamoyl group, it was thought worthwhile to study the
anticancer activity of the synthesized 1Hꢀbenzimidazole derivatives containing
cinnamoyl group against NCI 60 cell panel.
Further, the interactions of benzimidazole derivatives and compounds
containing cinnamoyl group with a biomolecule like HSA elucidated proper
understanding of biological processes such as absorption, transportation, distribution,
metabolism and excretion. It helps in designing new drug molecules. It is wellꢀknown
that transportation and disposition of endogenous and exogenous ligands present in
the blood are generally carried out by HSA.20 Further, it has the capacity of binding
reversibly to a large variety of small molecules that it serves as a carrier.21 The affinity
between a drug and HSA can alter the pharmacokinetics and pharmacodynamics of a
drug. The drug that has either too high or too low affinity towards HSA would be
rendered ineffective.22,23

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In our recent work, we have reported the synthesis, characterization, antiꢀ
tubercular and cytotoxicity of 1ꢀ[(2E)ꢀ3ꢀphenylpropꢀ2ꢀenoyl]ꢀ1Hꢀbenzimidazole
derivatives.24 Good results of preliminary studies encouraged us to investigate the
anticancer activity of 1Hꢀbenzimidazole derivatives against NCI 60 cell panel and to
carry out the interaction of the potent anticancer molecule with HSA.
Results and discussion
Synthesis and characterization of 1H-benzimidazoles
A series of 1ꢀ[(2E)ꢀ3ꢀphenylpropꢀ2ꢀenoyl]ꢀ1Hꢀbenzimidazole derivatives were
prepared as reported earlier (Fig. 1).24 In brief, slow addition of freshly prepared
cinnamoyl chloride to substituted benzimidazoles in dry THF in the presence of
º
sodium hydride at 0ꢀ5 C yielded 1ꢀ[(2E)ꢀ3ꢀphenylpropꢀ2ꢀenoyl]ꢀ1Hꢀbenzimidazole
1
derivatives. The synthesized molecules were characterized by spectral methods. H
NMR spectra of all the compounds exhibited two doublets each around 6.12 ꢀ 7.20
ppm and 7.57 ꢀ 8.05 ppm. These were attributed to vinylic protons and the coupling
constants of these protons (3JHH) at ca 16 Hz proved that these two protons were trans
to each other. Further, all the aromatic protons were observed in the expected region.
13
The C NMR spectra of all the compounds exhibited signals around 128.9 ꢀ 133.4
ppm and 145.5 ꢀ 149.2 ppm due to vinylic carbon atoms. The carbonyl carbon
appeared around 162.52 ꢀ 168.1 ppm and the aromatic carbon atoms were resonated in
the expected region. Further, single crystals of 1ꢀ[(2E)ꢀ3ꢀphenylpropꢀ2ꢀenoyl]ꢀ2ꢀ(4ꢀ
chlorophenyl)ꢀ1Hꢀbenzimidazole were grown and Xꢀray studies were carried out to
authenticate the structure.
Single crystal X-ray studies

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Single crystal of the compound 3f was developed from its alcoholic solution by
slow evaporation. The ORTEP diagram of 3f is presented in Fig. 2a. The developed
crystals belonged to monoclinic system with a space group of P21/c. The unit cell
parameters and crystal refinement25 data of the compound, 3f are given in Table 1.
Though there are no noteworthy hydrogen bonds in 3f molecules, its molecules
were stabilized by weak intermolecular CꢀH … N interactions in the solid state with a
distance of 2.653 Ǻ (Fig. 2b). The benzimidazole plane of 3f makes a dihedral angle
of 22.00 (3)º with cinnamoyl group due to steric hindrance of bulkier 4ꢀClꢀphenyl
ring. Further, 4ꢀClꢀphenyl ring has a dihedral angle of 49.90 (3)º with the mean plane
of benzimidazole ring system. Bond lengths and bond angles were observed to be in
normal ranges. The data on bond lengths and bond angles, coꢀordinates and
displacement parameters have been deposited at Cambridge Crystallographic Data
Centre (CCDC 1018656).
Anticancer activity
The synthesized compounds were submitted to National Cancer Institute (NCI),
Bethesda, Maryland, USA, under the Developmental Therapeutic Program (DTP) for
screening anticancer activity.26 The screening comprises 60 human tumor cell lines
(NCI60), representing leukemia, melanoma and cancers of the lung, colon, brain,
ovary, breast, prostate and kidney.27 The study involved twoꢀstage process; firstly, the
compounds were tested against 60 cell lines at a single dose concentration of 10ꢀ5 M.
The compounds that exhibited a significant growth inhibition were tested at five
concentration levels against the 60 cell panel. The compounds viz., 3a(NSC:
773399/1), 3b (NSC: 773400/1), 3c (NSC: 773401/1), 3d (NSC: 773402/1), 3e (NSC:

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773403/1), 3f (NSC: 773404/1), 3g (NSC: 773405/1) and 3h (NSC: 773406/1) were
subjected to full NCI 60 cell panel screening at a single dose (10ꢀ5 M).
Results of the single dose assessment are presented as a graph of mean growth
percent of the treated cells. These results revealed both growth inhibition (between 0
and 100 growth percent) and cytotoxicity values (with less than 0 growth percent).
The outputs from the single dose screening were analyzed by COMPARE program.28
The compounds 3a, 3d, 3e, 3g and 3h have displayed zero sensitivity while 3b and 3c
exhibited low to moderate sensitivity. 3f showed a considerable antiproliferative
activity towards human tumor cell lines. The significant percentage growth inhibition
(GI%) of the treated cells at 10ꢀ5 M concentrations of compounds are presented below:
Compound 1b (with 5,6ꢀdimethyl substituent on benzimidazole): Leukemia CCRFꢀ
CEM (GI% 64.66), RPMIꢀ8226 (GI% 63.61), SR (GI% 64.66),Breast CancerMCFꢀ7
(GI% 69.59); compound 1c(with 6ꢀmethyl substituent on benzimidazole): Leukemia
CCRFꢀCEM (GI% 60.81), RPMIꢀ8226 (GI% 88.17), SR (GI% 84.70), Colon Cancer
HCTꢀ116 (GI% 64.84), Breast Cancer MCFꢀ7 (GI% 73.43); compound 3f (with 4ꢀClꢀ
phenyl ring on C2ꢀbenzimidazole): Leukemia Kꢀ562 (GI% 60.22), MOLTꢀ4 (GI%
78.07), Non-Small Cell Lung Cancer NCIꢀH460 (GI% 99.46), Colon Cancer HCCꢀ
2998 (GI% 94.92), CNS Cancer SNBꢀ75 (GI% 81.93), Melanoma MALMEꢀ3M (GI%
84.12), Ovarian Cancer IGROV1 (GI% 89.62), OVCARꢀ4 (GI% 69.96), NCI/ADRꢀ
RES (GI% 89.22), SKꢀOVꢀ3 (GI% 76.05), Renal Cancer TKꢀ10 (GI% 81.42),
Prostate Cancer PCꢀ3 (GI% 93.91) DUꢀ145 (GI% 88.87) and Breast Cancer MCF7
(GI% 89.71), HS 578T (GI% 80.03). However, the compound 3f has showed a
remarkable cytotoxic activity along with growth inhibition against most of the cancer

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cell lines and corresponding results are depicted in Table 2. The compounds which
reduced the growth of the cell lines to 32% or less were considered as in vitro
active.29,30 Thus, 3f (NSC: 773404/1) satisfied preꢀdetermined threshold growth
inhibition criteria and so, was further selected for NCI full panel five dose assay at 10ꢀ
fold dilutions of five different concentrations.
Anticancer activity at five dose concentrations
Five dose screening was carried out by evaluating the compounds against 60 cell
lines representing 9 tumor subpanels at five different concentration levels (0.01, 0.1,
1, 10 and 100 µM). The results were expressed in terms of three response parameters
GI50, TGI and LC50. These were obtained by plotting the values of log concentration
versus % growth inhibition for all cell lines (Fig. 3). From Fig. 3, it is apparent that
the compound 3f completely inhibited the growth of ovarian cancer cell line
NCI/ADRꢀRES. Further, this compound exhibited good to moderate anticancer
activity against several examined cancer cell lines with GI50 values in the range of
0.38 ꢀ 3.13 µM. The acquired data (Table 3) revealed noticeable and remarkable
sensitivity profile towards leukemia SR, melanoma LOX IMVI, leukemia RPMIꢀ
8226, leukemia CCRFꢀCEM, ovarian cancer OVCARꢀ8, breast cancer MCFꢀ7, renal
cancer UOꢀ31, melanoma MDAꢀMBꢀ435, ovarian cancer OVCARꢀ3, leukemia
MOLTꢀ4, renal cancer A498, renal cancer ACHN, colon cancer HCTꢀ116, nonꢀsmall
cell lung cancer NCIꢀH522 and melanoma UACCꢀ62 with GI50 values of 0.38, 0.59,
0.61, 0.73, 0.75, 1.01, 1.10, 1.11, 1.11, 1.13, 1.17, 1.18, 1.21, 1.23 and 1.27 µM
respectively. From these results, it is evident that, the compound 3f unveiled
noteworthy sensitivity against leukemia, melanoma and renal cancer with average

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sensitivity of a particular subpanel 1.06, 1.47 and 1.51 µM respectively. However, the
criterion for selectivity of a compound depends upon the ratio obtained by dividing
the average sensitivity of all cell lines (full panel) MID (µM) by their individual
subpanel MID (µM). Ratios between 3 and 6 refer to moderate selectivity; ratios
greater than 6 indicate higher selectivity towards the corresponding cell line, while
compounds not meeting either of these criteria rated as nonꢀselective.31 In this context,
compound 3f was found to be nonꢀselective; however managed to display a good
antitumor activity against the nine tumor subpanels tested with selectivity ratios
ranging from 0.79 to 1.53 and 0.47 to 1.69 at GI50 and TGI levels, respectively.
Structure activity relationship
Chew et al.32 have recently demonstrated that α, βꢀunsaturated carbonyl moiety,
also known as the Michael acceptor, can inhibit thioredoxin reductase through
nucleophilic addition of glutathione cysteineꢀSH residues and can potentially be
exploited for use at different concentrations in chemotherapeutic and chemopreventive
strategies. With α, βꢀunsaturated carboxyl group, the synthesized benzimidazoles offer
Michaelꢀacceptor properties. Further, from the single dose results, it is clear that the
compounds carrying activating groups viz., methyl, methoxy, phenyl and phenol
groups on benzimidazole moiety did not enhance the anticancer activity while the
compound containing 4ꢀchlorophenyl group (3f) showed moderate to good
antiproliferative activity. This suggested that the compounds having an electronꢀ
withdrawing group in the paraꢀposition of the phenyl ring were found to be more
active to Michael addition.33 The presence of 4ꢀchloro phenyl ring on benzimidazole
exhibited a better activity compared to rest of the synthesized molecules. However, it

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is still preliminary to draw any conclusion that the Michael addition might be the only
mode of action of these compounds. Finally, it is proposed that further derivatization
of these compounds could result in obtaining more selective anticancer agents.
Interactions of compound 3f with human serum albumin
To understand the binding characteristics of the potent anticancer molecule 3f with
transport protein, HSA, we have employed fluorescence method.
Steady state fluorescence quenching studies
Fluorescence spectroscopy is the most suitable technique to investigate the
interaction of a bioactive compound with a biomolecule34 since it provides the
information concerning the structural changes in the biomolecule upon interaction.
We have investigated the interaction between the potent anticancer compound 3f and
serum albumin, HSA. HSA is the most abundant protein constituent of blood plasma
and has been used as a model protein for diverse biophysical and physicochemical
studies. It has a single tryptophan (Trp) residue (Trpꢀ214) located in the hydrophobic
cavity of the subdomain IIA of the soꢀcalled Sudlow’s binding site I.35 The intrinsic
fluorescence of Trp residues is extremely sensitive to its environment and can be used
to comprehend the binding of small molecules to protein.
Fluorescence quenching may be induced by a variety of molecular interactions
with a quencher molecule. In the present study, the emission spectra of HSA in the
presence and absence of increasing amounts of 3f were recorded to examine the
interaction between 3f and HSA. Fig. 4 represents the concentration dependent
quenching of protein fluorescence by 3f. It could be clearly seen that HSA showed a
strong emission band at 335 nm upon excitation at 280 nm. The compound 3f did not

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exhibit emission under the experimental conditions. Addition of 3f to HSA led to a
significant decrease in the fluorescence intensity besides the red shift in emission
wavelength thereby suggesting the changes in the environment around HSA.
Fluorescence quenching may be either dynamic or static or both. The dynamic
can be differentiated from the static quenching based on their dependence on
temperature. Thus, to propose the fluorescence quenching mechanism, the
fluorescence quenching titration was carried out at different temperatures (288, 298
and 308 K) and the results were analyzed using the SternꢀVolmer equation36 shown
below:
F0/F = 1 + KSV [Q] = 1 + Kqτ0 [Q]
(1)
where F and F0 are the fluorescence intensities with and without the quencher,
respectively, KSV is the SternꢀVolmer quenching constant, Kq is the biomolecular
quenching constant, τ0 is the average life time of biomolecule without the quencher
and [Q] is the concentration of the quencher. The SternꢀVolmer quenching constants
were obtained from the plot of F0/F versus [Q] and the corresponding results are
tabulated in Table 4. The SternꢀVolmer plots (Fig. 5) manifest the decreasing trend of
KSV with increase in temperature indicating the presence of static quenching
mechanism.
Analysis of binding equilibria
When small molecules bind independently to a set of equivalent sites on
macromolecules, it is possible to evaluate the binding constant and number of
molecules binding to protein using the following equation:37
log [(F0ꢀF)/F] = log K + n log [Q]
(2)

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where K is the binding constant and n is the number of molecules of 3f binding to
albumin molecule. The values of K and n were calculated from the plot of
log [(F0ꢀF)/F] versus log [Q] and the results are shown in Table 4. The magnitude of K
indicated the strong binding between 3f and HSA. Further, the values of K decreased
with increase in temperature indicating the decreased stability of 3f-HSA complex at
higher temperatures. It was evident from the values of n that one molecule of 3f bound
to one molecule of HSA.
Thermodynamics of interaction and nature of the binding mode
The nonꢀcovalent interactions between the ligand and protein may include
hydrophobic forces, electrostatic interactions, van der Waals interactions, hydrogen
bonds etc.38 The forces acting between small molecules and macromolecules can be
proposed based on thermodynamic parameters, enthalpy change (∆H°) and entropy
change (∆S°). Hence, to elucidate the mode of binding between 3f and HSA, the
temperatureꢀdependence of the binding constant of 3fꢀHSA was determined at
different temperatures (288, 298 and 308 K). The thermodynamic parameters are
evaluated using the following equation:
log K = − ∆H°/2.303RT + ∆S°/2.303R
(3)
The plot of log K versus 1/T (Fig. 6) yielded the values of ∆H° and ∆S° and were
found to be ꢀ132.0 kJmolꢀ1 and ꢀ356.1 JKꢀ1molꢀ1 respectively. The values of ∆G° were
calculated using the equation given below:
∆G°= −2.303RT log K
(4)
From the thermodynamic stance, ∆H° > 0 and ∆S° > 0 proposes the hydrophobic
interaction while ∆H° < 0 and ∆S° < 0 specifies the van der Waals forces and/or

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hydrogen bond formation; and ∆H° ≈ 0 and ∆S° > 0 reflects an electrostatic force.39
Negative values of ∆H° and ∆S° (Table 4) observed in the present study revealed the
presence of hydrogen bond and van der Waals forces in the binding process. Negative
values of ∆G° indicated the spontaneity of the interaction between 3f and HSA.
Site specific interaction
It is supposed that the compound that interacts to serum albumin binds at a series
of relatively wellꢀdefined regions on the protein. Sudlow et al.40 have proposed two
discrete sites for binding on the protein viz., site I and site II. It is supposed that site I
of albumin has the affinity for warfarin, phenylbutazone etc., while that of site II
shows preferential affinity for ibuprofen, flufenamic acid etc. Digitoxin binding is
recognized to be independent of sites I and II. Hence, competitive experiments were
carried out to determine the site specificity of 3f on HSA and their binding constants
were calculated to be 3.50x103, 5.63x103 and 5.73x103 Mꢀ1 for 3f-HSA with
phenylbutazone, ibuprofen and digitoxin respectively at 303 K. Significant decrease in
the binding constant of 3f-HSA in the absence of the site probe was found to be
5.68x103 at 303 K. Decreased binding constant of 3f-HSA in the presence of
phenylbutazone suggested that the phenylbutazone was significantly displaced by
compound 3f. In other words, both of them compete for the same binding site in the
protein. So, the site I located in subdomain II A was proposed to be the core binding
site for 3f in HSA.
Effects of common ions on binding of 3f-HSA
Some common ions are extensively dispersed in humans and animals, which can
affect the interactions of compounds with serum albumin. So, metal ions may affect

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the interactions of a drug with HSA and thus it may influence the distribution,
pharmacological property and metabolism of medicine in the blood. In view of this,
the binding of 3f with HSA was investigated in the presence of common ions viz.,
Co2+, Cu2+, K+, Mn2+, Ni2+ and Zn2+ at 303 K. The corresponding binding constants
shown in Table 5 revealed that the binding constant of 3f-HSA decreased to various
degrees in the presence of above metal ions. This indicated that the presence of above
ions reduced the binding constant of 3fꢀHSA thereby causing 3f to be rapidly cleared
from the blood. This might lead to the need for more doses of 3f to achieve the desired
pharmacological effect.41 Further, these results indicated that the quenching efficiency
of 3f was influenced by metal ions.
Conformational changes in secondary structure of the protein upon interaction
Absorption studies
The changes in the secondary structure of the protein and its complex formation
with a small molecule can be investigated by absorption studies. Thus, to explore the
changes in the environment around the protein structure, we have recorded absorption
spectra of HSA in the presence of increasing amounts of 3f (Fig. 7). It was evident
from Fig. 7 that the absorbance of HSA increased upon the addition of increasing
concentrations of 3f. Thus, the change in polarity around the Trp residue and the
change in peptide strand of HSA molecules was specified by the change in its λmax and
hence the change in hydrophobicity. This represents the binding between 3f and
protein that led to changes in the conformation of the protein.

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3D fluorescence spectra
The maximum emission wavelength and fluorescence intensity of residues
showed a close relation to the polarity of their microenvironment. In 3Dꢀfluorescence
spectroscopy, the excitation wavelength, emission wavelength and fluorescence
intensity can be used as axes to investigate the integrated information of proteins.
Thus, to attain more information on the binding of 3f to HSA and to explore the
conformational changes in the protein, 3D spectra of HSA were recorded in the
presence and absence of 3f and the corresponding spectra are shown in Fig. 8a and 8b.
Peaks 1 and 2 (Fig. 8) are the Rayleigh scattering peak (λex = λem) and secondꢀ
ordered scattering peaks (λex = 2λem),42 respectively. In addition, peak A (λex/λem
=
230/316 nm) and peak B (λex/λem = 280/328 nm) were also observed in 3D
fluorescence spectra. Peak A is due to n→π* transition that is characteristic of the
polypeptide backbone structure43 of HSA while peak B represents the spectral
characteristics of Trp and Tyrosine (Tyr) residues, since the protein predominantly
displays the intrinsic fluorescence of Trp and Tyr residues upon excitation at 280
nm.44 Analysis of Fig. 8 elucidated that the intensities of peaks A and B decreased to
different degrees upon the addition of 3f. The decrease in peak A demonstrated that
the structure of the peptide strands has changed upon interaction with 3f and that of
peak B revealed that the interaction of 3f with HSA induced some conformational
changes in HSA, which were related to the hydrophobic microenvironment near Trp
and Tyr residues. From these results, it could be inferred that HSA experienced a
slight unfolding of the polypeptide chain of protein and conformational changes in the
presence of 3f.

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Experimental Protocol
Synthesis of 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole derivatives
Reported method24 (Fig. 1) was followed to synthesize 1Hꢀbenzimidazole
derivatives that involve drop wise addition of freshly prepared cinnamoyl chloride (1
eq.) to a solution containing substituted benzimidazoles (1 eq.) and sodium hydride
(1.1 eq.) in THF at 0ꢀ5 °C. The reaction was carried out in an inert atmosphere. Then,
the reaction mixture was allowed to attain ambient temperature and the stirring was
continued. After the completion of the reaction, the volume of THF was reduced to
half in vacuo and poured to crushed ice and the pH was adjusted to neutral. The
separated solid was filtered, washed with water, dried and finally purification was
achieved on a silica gel (60ꢀ120 mesh) column chromatography using ethyl
acetate/hexane as the eluent. Characterization of synthesized molecules is given in
supplementary information file.
Anticancer screening methodology
The evaluation of anticancer activity was carried out at National Cancer
Institute (NCI) Bethesda, Maryland, USA, under the Developmental Therapeutic
Program (DTP).45 The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM Lꢀ
glutamine. For a typical screening experiment, cells were inoculated into 96 well
microtiter plates in 100 µL at plating densities ranging from 5,000 to 40,000
cells/wells depending on the doubling time of individual cell lines. After cell
inoculation, the microtiter plates were incubated at 37 ºC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two

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plates of each cell line were 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
were solubilized in dimethyl sulfoxide at 400ꢀfold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug addition, an aliquot of
frozen concentrate was thawed and diluted to twice the desired final maximum test
concentration with complete medium containing 50 µg mLꢀ1 gentamicin. Additional
four, 10ꢀfold or ½ log serial dilutions were made to provide a total of five drug
concentrations plus control. Aliquots of 100 µL of these different drug dilutions were
added to the appropriate microtiter wells containing 100 µL of medium, resulting in
the required final drug concentrations. Following the drug addition, the plates were
incubated for an additional 48 h at 37 °C, 5% CO2, 95% air and 100% relative
humidity. For adherent cells, the assay was terminated by the addition of cold TCA.
Cells were fixed in situ by the gentle addition of 50 µL of cold 50% (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 min at 4 ºC. The supernatant was
discarded, and the plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was
added to each well, and plates were incubated for 10 min at room temperature. After
staining, the unbound dye was removed by washing five times with 1% acetic acid
and the plates were air dried. Bound stain was subsequently solubilized with 10 mM
trizma base, and the absorbance was read on an automated plate reader at a
wavelength of 515 nm. For suspension cells, the methodology was the same except
that the assay was terminated by fixing settled cells at the bottom of the wells by
gently adding 50 µL of 80% TCA (final concentration, 16% TCA). Using the seven

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absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the
presence of drug at five concentration levels (Ti)], the percentage growth was
calculated at each of the drug concentration level. Percentage growth inhibition can be
calculated using the following equation:
[(TiꢀTz)/(CꢀTz)] x 100 for concentrations for which Ti ≥ Tz
[(TiꢀTz)/Tz] x 100 for concentrations for which Ti<Tz.
Three dose response parameters were calculated for each experimental agent.
Growth inhibition of 50% (GI50) was calculated using the equation
[(TiꢀTz)/(CꢀTz)] x 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. The drug concentration resulting in a total growth
inhibition (TGI) is calculated from Ti = Tz. The LC50 (concentration of drug resulting
in a 50% reduction in the measured protein at the end of the drug treatment as
compared to that at the beginning) indicating a net loss of cells following the
treatment is calculated from [(TiꢀTz)/Tz] x 100 = ꢀ50. Values were calculated for each
of these three parameters if the level of activity was reached. However, if the effect
was not reached or is exceeded, the value for that parameter was expressed as greater
or lesser than the maximum or minimum concentration tested.
Materials and methods for the interaction and conformational studies of HSA in
the presence of 3f
Steady state fluorescence spectra were performed on a Agilent technologies Cary
Eclipse fluorescence spectrometer equipped with a Xenon flash lamp source and a
Cary single cell peltier for temperature control. Absorbances were recorded on a Jasco

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Vꢀ670 UVꢀvis spectrophotometer equipped with a 1.0 cm quartz cell. For emission
spectra, excitation and emission slits with a band pass of 5.0 nm were used for all
measurements. The excitation wavelength was set at 280 nm for HSA and emission
spectra were recorded in the wavelength range of 285ꢀ500 nm.
HSA was purchased from SigmaꢀAldrich, India and used without further
purification. A stock solution of HSA was prepared by dissolving the appropriate
amount of HSA (250 µM) in phosphate buffer of pH 7.4 in Millipore water, while
those of synthesized compounds were prepared in 30% ethanol (25 µM). HSA
solution was stored at 4 ºC. All measurements were carried out at the physiological pH
of 7.4 using 0.1 M phosphate buffer. Fluorescence intensities were recorded by
keeping the concentration of protein fixed at 2.5 µM and varying that of compound 3f
in the range of 0.25ꢀ2.00 µM at 288, 298 and 308 K. Absorption spectra were
recorded in the range of 200ꢀ460 nm. The 3D fluorescence spectra were performed
under the following conditions: the emission wavelength was recorded between 200
and 550 nm and the initial excitation wavelength was set at 200 nm with an increment
of 10 nm up to 350 nm. Other scanning parameters were identical to those maintained
for steady state fluorescence studies.
Displacement experiments were performed using different site probes viz.,
phenylbutazone, ibuprofen and digitoxin for site I, II and III, respectively. For this, the
concentration of HSA and compound 3f was maintained similarly as that maintained
for steady state fluorescence measurements. The corresponding fluorescence
intensities were recorded.

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Fluorescence spectra of 3fꢀHSA were recorded in the presence of some cations
(at 0.25 µM) viz., K+, Co2+, Cu2+, Ni2+, Mn2+ and Zn2+ upon excitation at 280 nm.
Conclusions
The anticancer activity of synthesized molecules has been investigated against full
NCI 60 cell panel at NCI, USA. Compound 3f (NSC: 773404/1) was found to possess
a considerable antiproliferative activity towards the majority of the human tumor cell
lines and selected for five dose assays at 10ꢀfold dilutions of five different
concentrations. This compound was found to display a wide spectrum of antitumor
activity against the nine tumor subpanels tested with selectivity ratios ranging from
0.79 to 1.53 and from 0.47 to 1.69 at GI50 and TGI levels, respectively. Single crystals
of more potent anticancer active compound, 3f were grown and their crystal
parameters were studied. Further, in vitro studies on interactions of 3f with HSA were
carried out. Conformational changes in secondary structure of the protein upon
interaction with 3f have been studied by spectroscopic techniques. Static quenching
mechanism was observed in the binding of 3f to HSA. Based on site marker
competitive experiments, the compound 3f was proposed to bind to subdomain IIA in
HSA. The thermodynamic parameters revealed that the hydrogen bond and van der
Waals forces played a major role in the interaction between 3f and HSA. Absorption
and 3Dꢀfluorescence studies confirmed the conformational changes in the secondary
structure of protein upon interaction.
Acknowledgments
We are grateful to NCI, NIH, Bethesda, USA for selecting our samples for in
vitro anticancer analysis under DTP. We wish to thank the University Grants

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Commission (UGC), New Delhi [F. No. 14ꢀ3/2012 (NS/PE) dated 14ꢀ03ꢀ2012] for
providing financial support under “Antitumor activity: an integrated approach” a
focused area of UPE program. The authors also acknowledge the University Scientific
Instrumentation Centre, Karnatak University, Dharwad for single crystal Xꢀray data.

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Glossary
3D – Three dimensional
CCDC ꢀ Cambridge Crystallographic Data Centre
COMPARE ꢀ A computerized, pattern recognition algorithm which has considerable
utility in the evaluation and exploitation of data generated by the NCI
screen.
GI50 ꢀ The concentration for 50% of maximal growth inhibition of cell proliferation.
HPLC – High Performance Liquid Chromatography
HSA – Human Serum Albumin
LC50 ꢀ The concentration for killing 50% of disease cell (Lethal Concentration)
MID – Mid point
NCI – National Cancer Institute
NSC – National Service Centre
ORTEP ꢀ Oak Ridge Thermal Ellipsoid Plot
SAR – Structure Activity Relationship
SRB ꢀ Sulforhodamine B
TGI ꢀ The concentration for total growth inhibition of cell proliferation.
THF – Tetrahydrofuran
Trp – Tryptophan
Tyr – Tyrosine

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Table Captions
Table 1 Crystal and structure refinement data of 3f
Table 2 One dose mean graph of nine different cancer cell types of compound 3f
(NSC: 773404/1).
Table 3 NCI in vitro testing results of compound 3f (NSC: 773404/1) at five dose
level in µM.
Table 4 Binding and thermodynamic parameters of 3fꢀHSA system.
Table 5 Effect of common ions on the binding of 3f to HSA at 303 K.

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Table 1 Crystal and structure refinement data of 3f
Parameter
Empirical formula
Formula weight
Temperature
C22H15ClN2O
358.82
296 K
Crystal size
0.30x0.20x0.20 mm
Colorless
Crystal color
Crystal system
Space group
Monoclinc
P21/c
a = 13.5275(10) Å,
b = 7.5746(5) Å,
c = 18.3908(11) Å,
α = 90°
Unit cell dimensions
β = 111.098(2)°
γ = 90°
Limiting indices
ꢀ15<=h<=14, ꢀ8<=k<=8, ꢀ20<=l<=20
Volume
1758.1 (2) Å3
Z, Calculated density
Reflections collected
No. of independent reflections
Data/Restraints/Paramaeters
Final R indices [I> 2σ(I)]
R indices (all data)
4, 1.359 mg/m3
10366
2764 [R(int) = 0.0330]
2764 / 0 / 236
R1 = 0.0395, wR2 = 0.1016
R1 = 0.0591, wR2 = 0.1159
Bruker SMART APEX2 CCD
Measurements
Diffractometer
SHELXLꢀ97 (Sheldrick, 2008)
Fullꢀmatrix least squares on F2
Refinement program
Refinement method
Goodness of fit
1.008
CCDC deposit number
CCDC 1018656

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Table 2 One dose mean graph of nine different cancer cell types of compound 3f (NSC: 773404/1).
Panel
Cell lines
Growth percent
Growth inhibition
Leukemia
CCRFꢀCEM
HLꢀ60 (TB)
Kꢀ562
MOLTꢀ4
RPMIꢀ8226
ꢀ23.88
ꢀ3.97
39.78
21.93
ꢀ31.13
Cytotoxic
Cytotoxic
60.22
78.07
Cytotoxic
Non-Small Cell Lung
Cancer
A549/ATCC
ꢀ4.99
Cytotoxic
HOPꢀ62
HOPꢀ92
ꢀ48.81
ꢀ39.42
ꢀ55.28
ꢀ24.46
45.05
0.54
ꢀ72.37
ꢀ60.78
5.08
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
54.95
NCIꢀH226
NCIꢀH23
NCIꢀH322M
NCIꢀH460
NCIꢀH522
COLO 205
HCCꢀ2998
HCTꢀ116
HCTꢀ15
99.46
Cytotoxic
Cytotoxic
94.92
Colon Cancer
ꢀ71.46
ꢀ24.78
ꢀ40.61
ꢀ62.91
ꢀ55.08
ꢀ11.85
ꢀ51.99
ꢀ48.44
41.12
18.07
ꢀ0.39
ꢀ82.92
15.88
ꢀ37.34
ꢀ44.30
ꢀ36.05
ꢀ65.41
ꢀ70.59
ꢀ64.42
ꢀ84.68
10.38
ꢀ57.18
30.04
ꢀ48.09
ꢀ31.30
10.78
23.95
ꢀ17.65
ꢀ14.61
ꢀ49.87
ꢀ39.45
ꢀ65.21
ꢀ48.49
18.58
ꢀ38.98
6.09
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
58.88
HT29
KM12
SWꢀ620
SFꢀ268
SFꢀ295
SFꢀ539
SNBꢀ19
SNBꢀ75
CNS Cancer
Melanoma
81.93
U251
Cytotoxic
Cytotoxic
84.12
LOX IMVI
MALMEꢀ3M
M14
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
89.62
Cytotoxic
69.96
Cytotoxic
Cytotoxic
89.22
MDAꢀMBꢀ435
SKꢀMELꢀ2
SKꢀMELꢀ28
SKꢀMELꢀ5
UACCꢀ257
UACCꢀ62
IGROVꢀ1
OVCARꢀ3
OVCARꢀ4
OVCARꢀ5
OVCARꢀ8
NCI/ADRꢀRES
SKꢀOVꢀ3
786ꢀ0
Ovarian Cancer
Renal Cancer
76.05
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
Cytotoxic
81.42
A498
ACHN
CAKIꢀ1
RXFꢀ393
SN12C
TKꢀ10
UOꢀ31
PCꢀ3
Cytotoxic
93.91
Prostate Cancer
Breast Cancer
DUꢀ145
MCFꢀ7
MDAꢀMBꢀ231/ATCC
HS 578T
BTꢀ549
11.13
10.29
ꢀ26.52
19.97
ꢀ50.47
ꢀ4.25
88.87
89.71
Cytotoxic
80.03
Cytotoxic
Cytotoxic
Cytotoxic
Tꢀ47D
MDAꢀMBꢀ468
ꢀ6.61

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Table 3 NCI in vitro testing results of compound 3f (NSC: 773404/1) at five dose level in µM.
Panel
Cell lines
GI50
TGI
LC50
Conc. Sub
Selectivity Conc.
Sub
Selectivity
ratio (MIDc
: MIDd)
Per
Cell
line
Panel
ratio
Per
Cell
line
3.45
4.88
11.8
6.73
3.82
1.88
7.30
Panel
MIDb
(MIDa :
MIDb)
1.53
MIDd
Leukemia
CCRFꢀCEM
HLꢀ60 (TB)
Kꢀ562
MOLTꢀ4
RPMIꢀ8226
SR
0.73
1.40
2.09
1.13
0.61
0.38
1.89
1.06
5.43
4.98
1.13
>100
>100
>100
>100
>100
>100
35.1
Non-Small
Cell Lung
Cancer
A549/ATCC
1.86
0.87
1.23
HOPꢀ62
HOPꢀ92
2.72
1.46
2.17
1.91
1.96
1.53
1.23
1.41
5.17
3.82
6.00
4.89
6.52
3.50
2.64
2.78
9.81
10.0
>100
20.0
25.5
8.04
5.65
5.49
NCIꢀH226
NCIꢀH23
NCIꢀH322M
NCIꢀH460
NCIꢀH522
COLO 205
Colon
Cancer
1.45
1.12
3.98
1.54
HCCꢀ2998
HCTꢀ116
HCTꢀ15
HT29
KM12
SWꢀ620
SFꢀ268
SFꢀ295
SFꢀ539
1.80
1.21
1.34
1.51
1.44
1.45
2.10
2.50
1.47
3.13
1.44
1.60
0.59
1.63
1.90
1.11
2.10
1.49
1.64
1.47
1.27
1.95
3.45
2.88
7.05
3.71
2.93
5.03
6.14
6.46
2.92
12.8
3.77
3.22
1.91
5.74
5.05
3.04
4.35
3.22
3.12
3.42
2.88
4.63
6.61
6.90
29.8
9.16
5.97
27.9
42.5
26.1
5.79
40.5
9.91
6.50
4.77
26.0
19.6
8.32
9.02
6.92
5.93
7.93
6.53
13.4
CNS Cancer
Melanoma
2.04
1.47
0.79
1.10
5.89
3.63
1.04
1.69
SNBꢀ19
SNBꢀ75
U251
LOX IMVI
MALMEꢀ3M
M14
MDAꢀMBꢀ435
SKꢀMELꢀ2
SKꢀMELꢀ28
SKꢀMELꢀ5
UACCꢀ257
UACCꢀ62
IGROVꢀ1
Ovarian
Cancer
1.78
1.51
0.91
1.07
12.92
0.47
1.14
OVCARꢀ3
OVCARꢀ4
OVCARꢀ5
OVCARꢀ8
NCI/ADRꢀRES
SKꢀOVꢀ3
786ꢀ0
1.11
3.01
1.74
0.75
1.49
2.43
2.05
2.64
1.67
4.21
10.3
61.3
5.71
7.63
6.29
49.7
10.5
62.1
>100
18.0
28.4
Renal
Cancer
5.36