Synthesis, spectroscopic characterization, crystal structure, DNA interaction study and in vitro biological screenings of 4-(5-chloro-2- hydroxyphenylamino)-4-oxobut-2-enoic acid

Article abstract of DOI:10.1016/j.saa.2014.06.099

The titled compound, 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid was synthesized and characterized by various techniques like elemental analyses, FT-IR, NMR (¹H, and ¹³C) and single crystal X-ray structural analysis. The appearance of the OH peak of the carboxylic acid in the FT-IR and NMR spectra conform the formation of the compound. A good agreement was found between the calculated values of C, H, N and found values in elemental analysis that show the purity of the compound. Protons H2 and H3 are in cis conformation with each other as conformed both from ¹H NMR as well as from single crystal X-ray analysis. The molecular structure of the title compound, C₁₀H₁₀NO₃Cl, is stabilized by short intramolecular OH- - -O hydrogen bonds within the molecule. In the crystal structure, intermolecular NH- - -O hydrogen bonds link molecules into zigzag chains resulting in a dendrimer like structure. The title compound was screened for biological activities like interaction with DNA, cytotoxicity, antitumor and antioxidant activities. DNA interaction study reveals that the binding mode of interaction of the compound with SS-DNA is intercalative as it results in hypochromism along with significant red shift of 5 nm. It was also found to be effective antioxidant of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and show almost comparable antioxidant activity to that of the standard and known antioxidant, ascorbic acid, at higher concentration. The antitumor activity data of the compound shows that it can be used as potent antitumor agent.

Full text of DOI:10.1016/j.saa.2014.06.099

Accepted Manuscript
Synthesis, Spectroscopic Characterization, Crystal Structure, DNA Interaction
Study and in Vitro Biological Screenings of 4-(5-chloro-2-hydroxyphenylami-
no)-4-oxobut-2-enoic acid
Muhammad Sirajuddin, Nooruddin, Saqib Ali, Vickie McKee, Shahan Zeb
Khan, Khan Malook
PII:
S1386-1425(14)00998-6
http://dx.doi.org/10.1016/j.saa.2014.06.099
SAA 12358
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2014
14 May 2014
16 June 2014
Please cite this article as: M. Sirajuddin, Nooruddin, S. Ali, V. McKee, S.Z. Khan, K. Malook, Synthesis,
Spectroscopic Characterization, Crystal Structure, DNA Interaction Study and in Vitro Biological Screenings of 4-
(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.06.099
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Synthesis, Spectroscopic Characterization, Crystal Structure, DNA Interaction Study and
in Vitro Biological Screenings of 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid
Muhammad Sirajuddina, Nooruddina, Saqib Alia,*, Vickie McKeeb, Shahan Zeb Khana,c,
Khan Malookd
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Department of Chemistry, Loughborough University, LE11 3TU, UK
c Department of Chemistry, University of Science and technology, Bannu, Pakistan
d Centralized Research Laboratory (CRL), University of Peshawar, K.P.K., Pakistan
*Corresponding author addresses:
drsa54@yahoo.com; m.siraj09@yahoo.com; Tel.: +92-51-90642130; Fax: +92-51-90642241.
Abstract
The titled compound, 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid was
synthesized and characterized by various techniques like elemental analyses, FT-IR, NMR (1H,
and 13C) and single crystal X-ray structural analysis. The appearance of the OH peak of the
carboxylic acid in the FT-IR and NMR spectra conform the formation of the compound. A good
agreement was found between the calculated values of C, H, N and found values in elemental
analysis that show the purity of the compound. Protons H2 and H3 are in cis conformation with
each other as conformed both from 1H NMR as well as from single crystal X-ray analysis. The
molecular structure of the title compound, C10H10NO3Cl, is stabilized by short intramolecular O-
H---O hydrogen bonds within the molecule. In the crystal structure, intermolecular N-H---O
hydrogen bonds link molecules into zigzag chains resulting in a dendrimer like structure. The
title compound was screened for biological activities like interaction with DNA, cytotoxicity,
antitumor and antioxidant activities. DNA interaction study reveals that the binding mode of
interaction of the compound with SS-DNA is intercalative as it results in hypochromism along
with significant red shift of 5 nm. It was also found to be effective antioxidant of 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH) and show almost comparable antioxidant activity to that of the
standard and known antioxidant, ascorbic acid, at higher concentration. The antitumor activity
data of the compound shows that it can be used as potent antitumor agent.
Keywords: Crystal structure; Cytotoxicity; DNA interaction; Antitumor activity; Antioxidant
activity
1. Introduction

Carboxylic acids have a variety of applications in industries producing food and pharmaceutical
products, surfactants, detergents, and green solvent. Currently, carboxylic acids have become
interesting as a raw material for biodegradable polymers. Plus the rise in petroleum costs, the
large-volume production of carboxylic acids by fermentation has been the focus of new attention
[1]. In the production of carboxylic acids by fermentation, free acid is required for the
manufacture of commodities and specialty chemicals. Moreover, contaminating proteins and cell
by-products need to be removed from the final product. Thus, to be cost effective, the separation
process requires the removal of cells and protein-like impurities, concentration of salt,
conversion of acid salts into free acids, and polishing of free acid to its required purity [2]. As
such, the costs associated with product recovery, concentration, acidification, and purification
have been very high in the past accounting for 60-70% of the product cost and making
fermentation-based chemical technology impractical account for 60-70% of the product cost [3].
In recent times, free – radicals are now recognized in the pathogenesis of a growing number of
diseases. Antioxidants are chemicals that offer up their own electrons to the free radicals, thus
preventing cellular damage. Many studies have shown that phytochemicals with antioxidant
activity may reduce the risk of cancer and heart disease. Continuous intake of antioxidants is
therefore necessary and important to reduce cellular damage and ageing process [4,5].
In this paper we are reporting the synthesis, characterization, crystal structural and biological
screening of a novel compound, 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid.
2. Experimental section
2.1. Materials and methods
Reagents 5-chloro-2-hydroxyanaline and maleic anhydride were obtained from Aldrich (USA)
and were used without further purification. All the solvents purchased from E. Merck (Germany)
were dried before use according to the literature procedures [6]. The melting points were
determined in a capillary tube using a Gallenkamp (U.K) electrothermal melting point
-1
apparatus. IR spectra in the range of
4000-400 cm were obtained on a Thermo Nicolet-6700
Elemental analysis was done using a CE-440 Elemental Analyzer
FT-IR Spectrophotometer.
(Exeter Analytical, Inc) and the experimentally found values are given in parenthesis in
1
experimental part. H, and 13C NMR were recorded on a 400 MHz JEOL ECS instrument,
using DMSO as an internal reference [1H (DMSO-d6) = 2.50 and 13C (DMSO-d6) = 39.5 ppm].
Chemical shifts are given in ppm and coupling constants (J) values are given in Hz. The

multiplicities of signals in 1H NMR are given with chemical shifts; (s = singlet, d = doublet, dd =
doublet of a doublet). Crystal data was collected at 296 (2) K on a Bruker Apex II CCD
diffractometer. Details are given in Table 2. All the non-hydrogen atoms were refined using
anisotropic atomic displacement parameters, and hydrogen atoms bonded to carbon were inserted
at calculated positions using a riding model. Hydrogen atoms bonded to O or N were located
from difference maps and their coordinates refined. SHELXTL was used to solve and
SHELX2012 to refine the structures.
2.2. Brine shrimp lethality assay
The shrimp’s lethality bioassay was performed as developed by Michael et. al. [7], and later
modified by Sleet and Brendel [8]. It was based on the ability to kill laboratory-cultured Artemia
nauplii (brine shrimp). Doxorubicin was used as a standard drug. The following concentrations
were used: 20 ppm, 10 ppm, 5 ppm and 2.5 ppm.
Artificial sea water was prepared using sea salt 34 g/L under constant aeration for 48h at room
temperature. Brine shrimp (Artemiasalina) eggs (Ocean Star Inc., USA) were hatched in shallow
rectangular dish (22 x 32 cm) filled with prepared seawater, having a plastic separator of 2 mm
in which numerous holes were clamped to make two unequal sections in the dish. The eggs
(about 25 mg) were sprinkled in the larger section, which was darkened (covered with aluminum
foil) whereas the smaller section was illuminated. After 24h of hatching, phototropic nauplii
(brine shrimp larvae) were collected by pipette from the lightened side. From each stock solution
25 µL of the test compound was placed in glass vials (25 mL) and 2 mL sea water was added. 15
shrimps were transferred to each vial after counting against light background, using Pasteur
pipette, and volume was raised up to 5 mL. The vials were kept under illumination at room
temperature (25 to 28°C). After 24h of incubation, surviving shrimps were observed with the
help of 3x magnifying glasses. The experiment was performed in duplicate. In case, where
control death occurred, the data was corrected using Abbott’s formula [9]:
Then LD50 (Lethal Dose that killed 50% of shrimps) was calculated by using Finny software
[10].
2.3. Potato disc antitumor assay

Antitumor activity of the synthesized compounds was tested by performing modified potato disc
antitumor assay [11,12]. A 48h old bacterial culture of At-10 strain of Agrobacterium
tumefaciens, a Gram negative soil bacterium, was used in this experiment. This assay was
performed with inoculums at four concentrations of test samples (20 ppm, 10 ppm and 5 ppm),
containing bacterial culture and 1.5 mL autoclaved distilled water. Red-skinned potatoes were
surface sterilized in 0.1% HgCl2 solution, washed three times with autoclaved distilled water,
and potato discs of size 8 mm x 4 mm were prepared using a sterilized cork borer. Autoclaved
plain agar solution (1.5%) was poured into Petri plates and allowed to solidify. Ten discs were
placed on the agar surface of each Petri plate, and 50 µL of inoculum was placed on the surface
of each disc. The plates were sealed with parafilm to avoid contamination and moisture loss and
were incubated at 28°C in dark. The experiment was carried out in triplicate and in strict aseptic
conditions. After 21 days of incubation, discs were stained with Lugol's solution (10% KI and
5% I2) and tumors were counted on each disc using dissecting microscope. Percent tumor
inhibition was calculated by the following equation [12]:
2.4. DPPH scavenging assay
Scavenging effect on 2,2-diphenyl-1-picryhydrazyl (DPPH) The solution of 2,2-diphenyl-1-
picryhydrazyl (DPPH) radical was obtained by dissolving 3.94 mg of DPPH in 100 mL
methanol. To 2.8 mL of the methanolic solutions of DPPH, 0.2 mL of compound solution with
different concentration ranging from 10 to 150 mg/mL was added. After 10 min the decrease in
absorption was measured at 517 nm of DPPH using UV–Visible Spectrophotometer. The actual
decrease in absorption was measured against that of the control and the percentage inhibition was
calculated. The same experiment was carried out on ascorbic acid which is a known antioxidant.
All test and analysis were run in duplicate and the results obtained were averaged [14,15].
2.5. DNA Interaction Study Assay by UV-Visible Spectroscopy
SS-DNA (50 mg) was dissolved by overnight stirring in double deionized water (pH = 7.0) and
kept at 4°C. Doubly distilled water was used to prepare buffer (20 mM Phosphate buffer
(NaH2PO4-Na2HPO4), pH = 7.2). A solution of (SS-DNA) in the buffer gave a ratio of UV
absorbance at 260 and 280 nm (A260/A280) of 1.8, indicating that the DNA is sufficiently free of
protein [16,17]. The DNA concentration was determined via absorption spectroscopy using the

molar absorption coefficient of 6,600 M-1 cm-1 (260 nm) for SS-DNA [18,19] and was found to
be 1.4 ×10−4 M. The compound was dissolved in 70% ethanol at a concentration of 1 mM. The
UV absorption titrations were performed by keeping the concentration of the compound fixed
while varying the SS-DNA concentration. Equivalent solutions of SS-DNA were added to the
complex and reference solutions to eliminate the absorbance of DNA itself. Compound-DNA
solutions were allowed to incubate for about 10 min at room temperature before measurements
were made. Absorption spectra were recorded using cuvettes of 1 cm path length at room
temperature (25 ± 1°C).
2.6. Synthesis of 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid
Stoichiometric amounts of 5-chloro-2-hydroxyaniline and maleic anhydride were dissolved
separately in glacial acetic acid. The 5-chloro-2-hydroxyaniline solution was added slowly to the
solution of maleic anhydride and a precipitate appeared. The mixture was stirred at room
temperature for 12h [20] (Scheme 1). The precipitates were filtered, washed thoroughly with
distilled water to remove the unreacted precursors and air dried. Rod-like yellow single crystals
of the compound used in X-ray diffraction studies were grown in an acetone solution by slow
evaporation of solvent at room temperature.
Yield: 92%: M.p. 121-123˚C: Mol. Wt.: 241.5: Anal. Calc. for C10H8NClO4: C, 49.7 (49.3); H,
3.3 (3.8); N, 5.8 (5.6): IR (4000-100 cm-1): 3152 ν (OH); 3395 ν (NH); 1748 ν (amide C=O);
1
1591 ν (COasym); 1350 ν (COsym); 241 (Δν); H NMR (DMSO-d6, 400 MHz) δ (ppm): 13.12
(s, 1H; COOH); 6.37-6.33 (d, 1H, H2, 3J[1H, 1H] = 12.0 Hz); 6.65-6.61 (d, 2H, H3, 3J[1H, 1H] =
4
1
12 Hz); 10.24 (s, 1H NH); 8.02-8.01 (d, 1H, H6, J[1H, H] = 2.4 Hz); 7.03-6.99 (dd, 1H, H8,
4J[1H, 1H] = 2.4 Hz); 6.90-6.87 (d, 1H, H9, 3J[1H, 1H] = 8.7 Hz); 9.87 (s, 1H, phenolic OH): 13C
NMR (DMSO-d6, 100 MHz) δ (ppm): 163.8 (C1); 130.9 (C2); 132.0 (C3); 167.8 (C4); 122.6
(C5); 121.9 (C6); 124.8 (C7); 127.4 (C8); 116.2 (C9); 147.2 (C10).

Scheme 1: Mechanism for the synthesis of 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-
enoic acid
3. Results and Discussion
3.1. FT- IR Spectra
FT-IR data reveal valuable information about the structures of the compound in the solid state.
The appearance of the OH peak at 3152 cm-1 in the FT-IR spectrum conform the formation of the
4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid. Similarly NH of the amide segment
gives a peak at 3395 cm-1 that also conform the formation of the compound. Infrared O-C=O
stretching frequencies have been utilized to discriminate coordinated from non-coordinated
carboxyl groups, and also, to recognize the nature of bonding of carboxylic group, namely,
monodentate or bridging. The value of Δν [Δν = νasym(COO) – νsym(COO)] for the reported
compound is 241 cm-1. The Δν value was also calculated from single crystal XRD data for
compound by using the following equation [21]:
where δr is difference between the two C-O bond lengths (Å) and θOCO is the O-C-O angle (°).
A good correlation was found between the Δν value calculated from FT-IR data (241) and single
crystal XRD data (244).
3.2. 1H and 13C NMR spectroscopy
1
13
The H and C NMR spectrum for the synthesized compound was recorded in DMSO using as
the external standard and the data are given in the experimental section. The numbering of the

1
compound is according to that shown in Scheme 1. The H NMR study of compound provides
further support for the formation of compound. The characteristic peak for the OH bond in the
spectrum of the compound at 13.12 ppm conform the formation of the compound. The NH gives
a singlet at 10.24 ppm. H2 and H3 appear as a doublets with 3J(1H-1H) coupling value of 12 Hz
that falls in the range of cis conformation. The phenyl proton at position 6 (H6) appears as a
doublet (d) at 8.02 ppm while the proton at position 8 (H8) appears as a doublet of doublets (dd)
at 7.03 ppm (HL). The proton at position 9 (H9) also appears as a doublet at 6.90 ppm.
13
1
The C NMR data of the ligand and their complexes were in agreement with the H NMR and
FT-IR data for the formation of complexes. The C=O resonance for C1 and C2 appeared at 163.8
and 167.8 ppm. However, the signals for –C-N-, and –CH=CH- peaks for the alkyl as well as
aryl groups appeared in their specific regions.
3.3. X-Ray Crystallography
Crystal data and structure refinement parameters for 4-(5-chloro-2-hydroxyphenylamino)-4-
oxobut-2-enoic acid are shown in Table 1 while the selected bond lengths and bond angles are
given in Table 2.
The title compounds, 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid crystallize as a
monomer in the space group of orthorhombic, Pnma. The molecular structures are shown in Fig.
1 while their unit cell packing is shown in Fig 2. The conformations of the N-H and the C=O
bonds in the amide segment are anti to each other. In the side chain, the amide C=O bond is anti
to the adjacent C-H bond, while the carboxyl C=O bond is syn to the adjacent C-H bond. The
hyrdogens of C1 and C2 are also syn to each other. The observed rare anti conformation of the
C=O and O-H bonds of the acid group is similar to that examined in N-(4-
Methoxyphenyl)maleamic acid [22-24]. There are intramolecular and intermolecular C-H---O
and N-H---O interactions that stabilize the packing structure of the compound. Intermolecular C-
H---O interaction links the chains with each other into a three-dimensional network via C-H---π
interactions. The details of intramolecular H-bonds observed in 4-(5-chloro-2-
hydroxyphenylamino)-4-oxobut-2-enoic acid are given in Table 3.
Figures 1 and 2
Tables 1-3
2.2. Cytotoxicity

The synthesized compound showed cytotoxic effect while using brine shrimp as a substrate.
Doxorubicin was used as a standard drug. The LD50 value of the investigated compound is 62.4
ppm. The data is shown in Table 4.
Table 4
2.3. DPPH Scavenging Activity
The model of the scavenging of the stable DPPH radical is extensively used to evaluate
antioxidant activities in less time than other methods. The reducing abilities of the was
determined by their interaction with the free stable radical 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) at different concentrations. The proposed mechanism of antioxidant activity is shown in
Scheme 2. Antioxidants can react with DPPH and produce 1,1-diphenyl-2-picryl-hydrazine. Due
to its odd electron DPPH gives a strong absorption band at 517 nm appearing a deep violet
colour [15]. As this electron becomes paired off in the presence of a free radical scavenger, the
absorption disappears and the resulting decolorization is stoichiometric with respect to the
number of electrons taken up. The change of absorbance produced in this reaction is reviewed to
evaluate the antioxidant potential of test samples and this assay is useful as a primary screening
system [15]. The DPPH radical is a stable free radical (due to extensive delocalization of the
unpaired electron) having λmax at 517 nm. When this compound abstracts a hydrogen radical
from compoun then absorption vanishes due to the absence of free electron delocalization [15].
The percent scavenging activity of compound was calculated as:
where As is the absorbance of the DPPH in the presence of the tested compound and Ao is the
absorbance of the DPPH in the absence of the tested compound (control).
The antioxidant activity of the compound is shown in Table 5 with comparison to standard
antioxidant ascorbic acid. The data show that with increase in concentration of the test
compound the intensity of radicals vanishes rapidly at a particular wavelength (517 nm). The
compound has scavenging activity that probably is due to the OH, NH group which can react
with DPPH radical by the typical H-abstraction reaction (HAT) to form a stable macromolecular
radical.

Scheme 2: The proposed mechanism of antioxidant activity
Table 5
2.4. Antitumor Activity
The antitumor activity of the newly synthesized compound was studied using the potato disc
bioassay technique. Minimum inhibitory concentration (MIC) of samples on Agrobacterium
tumefaciens is shown in Table 6. The exact mechanism of action is not till known but antitumor
properties of the compounds may be probably due to inhibition of mitochondrial oxidative
phosphorylation (the process of the synthesis of ATP by phosphorylation of ADP for which
energy is obtained by electron transport and which takes place in the mitochondria during
aerobic respiration) [25]. The Agrobacterium tumefaciens-induced potato disc tumor assay is an
effective indicator of antitumor activity regardless of the mechanism of drug action. Thus, this
assay would be acceptable as a primary general screen for antitumor activity of the synthesized
compounds, regardless of mode of inhibitory action on tumor formation. A pictorial presentation
of the antitumor activity assay is shown in Fig.3.
Table 6
Figure 3
2.5. DNA Interaction Study By UV-Visible Spectroscopy
Electronic absorption spectrum was initially used to examine the interaction between compound
and SS-DNA. Figure 4 shows the UV-visible spectrum of compound interaction with different
concentrations of DNA. It was observed that the compound has one strong absorption peak at
307.80 nm that is due to π-π interaction between the compound and the nitrogenous bases of the

DNA. After interaction with increasing amount of DNA, the peaks decreased gradually and the
wavelength has red shift of 5 nm. Long et al. [26] had pointed out that the absorption peaks shift
of the small molecules after they interacted with DNA could be as the clues to judge the binding
mode between the small molecules and DNA: If the binding involves a typical intercalative
mode, an hypochromism effect coupled with obvious bathochromism for the characteristic peaks
of the small molecules will be found due to the strong stacking between the chromophore and the
base pairs of DNA [27]. Therefore, based on this viewpoint, the interaction between compounds
(1-3) and SS-DNA could be noncovalent intercalative binding. After intercalating the base pairs
of DNA, the π* orbital of the intercalated ligand could couple with π orbital of base pairs, thus
decreasing the π-π* transition energy, and further resulting in the bathochromism. On the other
hand, the coupling of a π orbital with partially filled electrons decreases the transition
probabilities hence results hypochromic shift. Since hypochromism due to π-π* stacking
interactions may appear in the case of the intercalative binding mode, while bathochromism (red-
shift) may be observed when the DNA duplex is stabilized [27].
Based upon the variation in absorbance, the intrinsic binding constant of the compound with
DNA were determined according to Benesi-Hildebrand equation [28]:
A0
G
G
1



A A0 HG G HG G
K

DNA

Where K is the association/binding constant, A0 and A are the absorbances of the compound and
its complex with DNA, respectively, and G and HG are the absorption coefficients of the
compound and the compound-DNA complex, respectively. The association constants were
obtained from the intercept-to-slope ratios of A0/(A-A0) vs. 1/[DNA] plots. The Gibb's free
energy (ΔG) was determined from the equation:
ΔG = -RT lnK
where R is general gas constant (8.314 JK-1.mol-1) and T is the temperature (298 K).
The binding constant was found to be 3.8 x 103 M-1 while the Gibb's free energy was -20.4
KJ.mol-1. It can be concluded from the antitumor activity and DNA interaction study that the
synthesized compound might be used as a potent antitumor agent. DNA and enzymes represent
the most targeted bioreceptors for small molecules as various regulatory processes such as gene
expression, gene transcription, mutagenesis, carcinogenesis [29]. Most anticancer drugs bind to
DNA and proteins either in a reversible or irreversible manner suggesting a direct relationship

between their interactions with macromolecules, hence, leading to their therapeutic effect [30]. It
was proposed that the compounds interact with nitrogenous bases of nucleotides of nucleic acid
and inhibit the cell division by interfering the replication and transcription of DNA molecules.
The compounds may also affect the multienzyme complexes responsible for replication and
transcription of DNA thus causing a stop of proliferation of the cells [31]. From the interaction
study with SS-DNA it can be concluded that the synthesized compounds can be used as an
anticancer drug.
Figure 4
4. Supplementary material
Crystallographic data for 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid reported in
this paper has been deposited with the Cambridge Crystallographic Data Centre, CCDC#
978643. Copies of this information may be obtained free of charge from The Director, CCDC,
12, Union Road, Cambridge CB2 1EZ [Fax: +44 1223 336 033] or deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk.
Conclusions
Carboxylic acids are the example of compounds with wide industrial applications and high
potential. The compound, 4-(5-chloro-2-hydroxyphenylamino)-4-oxobut-2-enoic acid, was
characterized successfully by FT-IR, NMR, elemental analysis and single crystal structural
analysis. A good correlation was found between the Δν value calculated from FT-IR data and
single crystal XRD data. From the single crystal X-ray structural analysis it is revealed that the
compound has packing diagram like dendrimer with characteristics that makes them useful for
numerous biological applications. The synthesized compound was found an effective scavenging
of the stable DPPH radical DPPH as potent as standard antioxidant agent, ascorbic acid. The
ability of the synthesized compound to be used as primary antioxidant is thus significant. It also
exhibit cytotoxic activity against brine shrimp. The compound interacts with DNA via
intercalation mode of interaction in which the compound inserts itself into the base pair of DNA.
The binding mode for interaction of compound with the SS-DNA was conformed by UV-visible
spectroscopic technique.
Acknowledgment
The author, Muhammad Sirajuddin, gratefully acknowledge the Higher Education Commission
(HEC) Islamabad, Pakistan, for financial support.

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Figures captions:
Fig. 1: A perspective view of the compound showing the crystallographic numbering scheme,
with displacement ellipsoids drawn at the 50% probability level.
Fig. 2: Packing digram with unit cell of compound viewed along a-axis.
Fig. 3: Antitumor activity of the compound with IC50 = 0.4.
Fig. 4: Absorption spectrum of 1 mM compound in the absence (a) and presence of 20 (b), 40
(c), 60 (d), 80 (e), 100 (f), 120 (g), 140 (h), 160 (i) and 180 (j) µM DNA. The arrow indicates the
increasing conc. of DNA. The inset graph represents the plot of Ao/A-Ao vs. 1/[DNA] (µM)-1 for
the calculation of binding constant (K) and Gibb’s free energy (∆G).
Fig. 1

Fig. 2
Fig. 3

Fig. 4

Tables:
Table 1: Crystal data and structure refinement parameters
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
C10H8ClNO4
241.62
296(2)
orthorhombic
Pnma
a/Å
10.7248(11)
b/Å
6.5805(6)
c/Å
14.3040(15)
α/°
90
β/°
90
γ/°
90
Volume/Å3
1009.50(17)
Z
4
ρcalcmg/mm3
1.590
m/mm-1
0.375
(000)
496.0
Crystal size/mm3
0.12 × 0.09 × 0.06
MoKα (λ = 0.71073)
4.748 to 54.212°
-13 ≤ h ≤ 9, -8 ≤ k ≤ 7, -16 ≤ l ≤ 18
3538
Radiation
2Θ range for data collection
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
1189 [Rint = 0.0192, Rsigma = 0.0210]
1189/0/97
1.052
R1 = 0.0388, wR2 = 0.1007
R1 = 0.0479, wR2 = 0.1078
0.25/-0.32

Table 2: Bond lengths and Bong angles
Bond Lengths (Å)
O1—C1
C1—O2
C1—C2
C2—C3
C3—C4
C4—O3
C4—N1
N1—C5
1.308 (3)
1.217 (3)
1.488 (3)
1.325 (3)
1.478 (3)
1.239 (3)
1.337 (3)
1.410 (3)
C5—C6
C5—C10
1.393 (3)
1.394 (3)
1.382 (3)
1.371 (4)
1.741 (2)
1.387 (3)
1.383 (3)
1.359 (3)
C6—C7
C7—C8
C7—Cl1
C8—C9
C9—C10
C10—O4
Bond angles (º)
O2—C1—O1
O2—C1—C2
O1—C1—C2
C3—C2—C1
C2—C3—C4
O3—C4—N1
O3—C4—C3
N1—C4—C3
C4—N1—C5
C6—C5—C10
C6—C5—N1
120.7 (2)
119.1 (2)
120.23 (19)
132.3 (2)
128.7 (2)
121.8 (2)
123.8 (2)
114.35 (19)
129.47 (19)
120.1 (2)
124.1 (2)
C10—C5—N1
115.82 (19)
118.5 (2)
122.1 (2)
118.86 (19)
119.02 (19)
119.1 (2)
120.4 (2)
123.7 (2)
116.5 (2)
119.8 (2)
C7—C6—C5
C8—C7—C6
C8—C7—Cl1
C6—C7—Cl1
C7—C8—C9
C10—C9—C8
O4—C10—C9
O4—C10—C5
C9—C10—C5
Table 3: Hydrogen-bond geometry (Å, º)
D—H···A
O1—H1A···O3 0.82
C2—H2···O4i
0.93
C3—H3···O2ii 0.93
C6—H6···O3 0.93
D—H
H···A
1.67
2.50
2.43
2.26
D···A
D—H···A
2.488 (2)
3.378 (3)
3.352 (3)
2.845 (3)
177
157
169
121
Symmetry codes: (i) x+1/2, -y+1/2, -z+3/2; (ii) x-1/2, -y+1/2, -z+3/2.

Table 4: Brine Shrimp Cytotoxicity Assay Results
Total No. of
Shrimps at
each dose level
30
No. of Shrimps killed at each dose level
LD50
(ppm)b
20 ppm
10 ppm
5 ppm
2.5 ppm
13
30
11
27
10
19
9
62.4
2.8
Compound
Doxorubicina
30
15
a) Positive control
b) LD 50= Lethality dose at which 50 % shrimps are killed
Table 5: DPPH Free Radical Scavenging Assay
Percentage Scavenging
IC50
200
66.6
22.2
7.4
µg/mL
2.4
µg/mL
µg/mL
90.071
91.735
µg/mL
78.799
88.155
µg/mL
51.659
82.035
µg/mL
43.478
63.860
44.381
81.893
10.5
1.9
Compound
Ascorbic acida
a) Positive control
Table 6: Potato disc antitumor data
20 ppm
Dose level
10 ppm
5 ppm
96.6
IC50 ppm
70
82
86
90
Compound
Doxorubicina
0.4
-
99.6
a) Positive control

Graphical Abstract
Synopsis for graphical abstract
The titled compound makes a dendrimer like packing structure vied along a-axis. The dendrimers
are very important on pharmaceutical side as act as a wonderful tool for the drug delivery
system. It inserts into the base pairs of DNA while interacting via intercalative mode.

Highlights
• Synthesis of novel compound
• Structural and spectroscopic characterization
• Biological activities
• Interaction with SSDNA via intercalative mode of interaction
• Excellent Antioxidant activity