Potential bioactive Schiff base compounds: Synthesis, characterization, X-ray structures, biological screenings and interaction with Salmon sperm DNA

Article abstract of DOI:10.1016/j.saa.2013.06.096

(Figure Presented) Three Schiff base compounds of N′-substituted benzohydrazide and sulfonohydrazide derivatives: N′-(2-hydroxy-3- methoxybenzylidene)-4-tert-butyl- benzohydrazide (1), N′-(5-bromo-2- hydroxybenzylidene)-4-tert-butylbenzohydrazide (2) and

Full text of DOI:10.1016/j.saa.2013.06.096

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Potential bioactive Schiff base compounds: Synthesis, characterization,
X-ray structures, biological screenings and interaction with Salmon
sperm DNA
b
Muhammad Sirajuddin a, Noor Uddin a, Saqib Ali a, , Muhammad Nawaz Tahir
⇑
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Department of Physics, University of Sargodha, Pakistan
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Three Schiff bases of N0-substituted benzohydrazide derivatives were synthesized and characterized suc-
cessfully. All the synthesized compounds interact with SS-DNA via intercalation mode of interaction.
ꢀ
ꢀ
Synthesis of potential bioactive Schiff
base compounds.
Structural and spectroscopic
characterization.
ꢀ
ꢀ
Biological activities.
Interaction with SSDNA via
intercalative mode of interaction.
Excellent antioxidant activity.
ꢀ
a r t i c l e i n f o
a b s t r a c t
Three Schiff base compounds of N0-substituted benzohydrazide and sulfonohydrazide derivatives:
N0-(2-hydroxy-3-methoxybenzylidene)-4-tert-butyl- benzohydrazide (1), N0-(5-bromo-2-hydroxyben-
zylidene)-4-tert-butylbenzohydrazide (2) and N0-(2-hydroxy-3-methoxybenzylidene)-4-methyl-
benzenesulfonohydrazide (3) were synthesized and characterized by elemental analysis, FT-IR, 1H,
13C NMR spectroscopy and single crystal analysis. The title compounds have been screened for their bio-
logical activities including, antibacterial, antifungal, antioxidant, cytotoxic, enzymatic activities as well as
interaction with SS-DNA which showed remarkable activities in each area of research. The DNA binding
of the compounds 1–3 with SS-DNA has been carried out with absorption spectroscopy, which reveals the
binding propensity towards SS-DNA via intercalation mode of interaction. The intercalative mode of
interaction is also supported by viscometric results. The synthesized compounds were also found to be
effective against alkaline phosphatase enzyme. They also show significant to good antimicrobial activity
against six bacterial and five fungal strains. The MIC (minimum inhibitory concentration) for antibacterial
Article history:
Received 11 February 2013
Received in revised form 24 June 2013
Accepted 27 June 2013
Available online 16 July 2013
Keywords:
Schiff base
Single crystal
DNA interaction
Antimicrobial activity
Enzymatic activity
Antioxidant activity
activity ranges from 1.95–500
lg/mL. Compounds 1–3 show cytotoxic activity comparable to the control.
At higher conc. (100 g/L) compound 3 shows 100% activity means that it has killed all brine shrimps.
l
They were 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.
Ó 2013 Elsevier B.V. All rights reserved.
⇑
Corresponding author. Tel.: +92 51 90642130; fax: +92 51 90642241.
E-mail address: drsa54@yahoo.com (S. Ali).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.06.096

112
M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
NMR, X-ray single crystal technique and elemental analyses. Melt-
Introduction
ing points were determined in a capillary tube using a Gallenkamp
(UK) electrothermal melting point apparatus. IR spectrum in the
range of 4000–400 cmꢁ1 was obtained on a Thermo Nicolet-6700
FT-IR Spectrophotometer equipped with DTGS (deuterated trigly-
cine sulphate) detector. Microanalysis was done using a Leco CHNS
932 apparatus. 1H and 13C NMR were recorded on a Bruker-
300 MHz FT-NMR Spectrometer, using CDCl3 as an internal refer-
ence [1H (CDCl3) = 7.25 and 13C (CDCl3) = 77] [17]. 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, t = triplet, m = multiplet). The
absorption spectrum was measured on a Shimadzu 1700 UV–Visi-
ble Spectrophotometer. The electrical conductance was measured
on a GENWAY 4510 Conductivity Meter. The X-ray diffraction data
were collected on a Bruker SMART APEX CCD diffractometer,
equipped with a 4 K CCD detector set 60.0 mm from the crystal.
The crystals were cooled to 100 1 K using the Bruker KRYOFLEX
low temperature device and Intensity measurements were per-
Schiff bases (azomethines) represent one of the most widely
used classes of organic compounds, not only as synthetic interme-
diates but also in coordination chemistry [1]. Schiff bases derived
from an amino and carbonyl compound are an important class of
ligands that coordinate to metal ions via azomethine nitrogen
and have been studied extensively. In Schiff base derivatives, the
C@N linkage is vital for biological activity, several Schiff bases were
reported to possess notable antibacterial, antifungal, anticancer
and diuretic activities. Schiff bases have wide applications in food
industry, dye industry, analytical chemistry, catalysis, fungicidal,
agrochemical and biological activities [2–4]. Schiff bases of gossy-
pol show high antiviral activity [5]. Schiff base derived from sul-
fane thiadizole and salicylaldehyde or thiophene-2-aldehydes
and their complexes show toxicities against insects [6]. a-Amino-
acid acts as intermediate in synthesis of photostable pyrthriod
insecticides [7].
DNA is one of the most important biomacromolecules in life
processes because it carries inheritance information and instructs
the biological synthesis of proteins and enzyme through the pro-
cess of replication and transcription of genetic information. It plays
an important role in the process of storing, copying and transmit-
ting gene messages. DNA is also a major target for drugs and some
harmful chemicals, and the studies on the binding nature of these
small molecules to DNA are important and fundamental issues on
life science because these drugs and chemicals can significantly
influence the genetic information expression and result in some
diseases related to the cell proliferation and differentiation [8,9].
Generally, the small molecules interact with DNA via three kinds
of noncovalent modes, i.e., (i) intercalating between stacked base
pairs, (ii) groove binding, or (iii) electrostatic bind to the negatively
charged nucleic acid sugarphosphate backbone [10,11]. Intercala-
tion involves the insertion of a planar molecule between DNA base
pairs, which results in a decrease in the DNA helical twist and
lengthening of the DNA [12]. Groove binding, unlike intercalation,
does not induce large conformational changes in DNA and may be
considered similar to standard lock-and-key models for ligand–
macromolecular binding. Groove binders are usually crescent-
shaped molecules that bind to the minor groove of DNA. They
are stabilized by intermolecular interactions and typically have lar-
formed using graphite monochromated Mo K
a radiation from a
sealed ceramic diffraction tube (SIEMENS). Generator settings were
50 kV/40 mA. The structure was solved by Patterson methods and
extension of the model was accomplished by direct methods using
the program DIRDIF or SIR2004. Final refinement on F2 carried out
by full-matrix least squares techniques using SHELXL-97, a modi-
fied version of the program PLUTO (preparation of illustrations)
and PLATON package.
Synthesis
Stoichiometric amounts of 4-tert-butylbenzohydrazide (for 1
and 2), 4-methylbenzenesulfonohydrazide (for 3) and 2-hydroxy-
3-methoxybenzaldehyde (for 1 and 3) 5-bromo-2-hydroxybenzal-
dehyde (for 2), (5 mmol of each) were added to freshly dried
toluene. The mixture was refluxed for 3–4 h and the water formed
was removed by using Dean and Stark apparatus. The reaction mix-
ture volume was reduced to one-third of its original and left for
crystallization at room temperature. The white crystals of 1 and
2 suitable for a single crystal analysis were isolated from the
mother liquor and dried. In case of 3, reddish yellow viscous liquid
was obtained that was solidified after two weeks. The chemical
reactions are shown in Scheme 1.
ger binding constants than intercalators (approximately 1011 Mꢁ1
)
[13]. The investigation of drug–DNA interactions is of current gen-
eral interest and importance [14,15] especially for the designing of
new DNA–targeted drugs and the screening of these in vitro.
In the present study we have synthesized three Schiff base
compounds:
Synthesis of N0-(2-hydroxy-3-methoxybenzylidene)-4-tert-butyl-
benzohydrazide (1)
Yield: 92%: m.p.: 150–151 °C: Mol. Wt.: 326.39: Anal. Calc. for
C
19H22N2O3: C, 69.92; H, 6.79; N, 8.58; Found: C, 69.98; H, 6.70;
N, 8.70%: IR (cmꢁ1): 1609, m (
C@N), 1425 and 1581, w ( C@C),
3271, br. ( OH), 1297, m ( NAC), 3480, m ( NAH stretch.), 1537,
w (
NAH bend.): 1H NMR (CDCl3, ppm): 1.35 (s, 9H, H1), 7.49–
N0-(2-hydroxy-3-methoxybenzylidene)-4-tert-butyl-benzohydrazide
(1), N0-(5-bromo-2-hydroxy-benzylidene)-4-tert-butylbenzohyd-
razide (2) and N0-(2-hydroxy-3-methoxybenzylidene)-4-methyl-
benzenesulfonohydrazide (3) were synthesized successfully and
characterized by various techniques. Biological activities including
DNA interaction, enzymatic study, antibacterial, antifungal, antiox-
idant and cytotoxic activities of the synthesized compounds were
studied.
m
m
m
m
m
m
7.52 (d, 2H, H4, H40, 3J[1HA1H] = 7.5 Hz), 7.83–7.85 (d, 2H, H5,
H50, 3J[1HA1H] = 7.5 Hz), 8.72 (s, 1H, H6), 6.93–6.97 (t, 1H, H9,
3J[1HA1H] = 12.3 Hz), 6.88–6.91 (m, 2H, H8 and H10), 3.95 (s,
H11), 10.80 (s, 1H, OH, NAHAO), 9.80 (s, 1H, NH); 13C NMR (CDCl3,
ppm): 35.2 (1C, C1), 31.3 (3C, C2), 155.3 (1C, C3), 125.8 (2C, C4,
C40), 128 (2C, C5, C50), 130.5 (1C, C6), 163.2 (1C, C7), 147.7 (1C,
C8), 119.4 (1C, C9), 121.4 (1C, C10), 119.5 (1C, C11), 114.2 (1C,
C12), 148.6 (1C, C13), 148.4 (1C, C14), 56.4 (1C, C15); Molar Con-
Materials and methods
Reagents, 4-methylbenzenesulfonohydrazide, 2-methoxy-6-methyl-
phenol, 4-tert-butylbenzo-hydrazide and 2,3-dihydroxybenzaldehyde
were obtained from Aldrich (USA), 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH) and used without further purification. Sodium salt
of Salmon fish sperm DNA (SS-DNA) (Arcos) was used as received.
All the solvents purchased from E. Merck (Germany) were dried
before used according to the literature procedure [16]. The
synthesized compounds were characterized by FT-IR, 1H and 13C
ductance in 70% ethanol (Km, l
S cm2 molꢁ1): 4.12 at 25 °C: Solu-
bility: chloroform, toluene, ethanol, methanol and DMSO.
Synthesis of N0-(5-bromo-2-hydroxybenzylidene)-4-tert-
butylbenzohydrazide (2)
Yield: 90%: m.p.: 170–171 °C: Mol. Wt.: 375.26: Anal. Calc. for
C18 H21 Br N2 O2: C, 57.61; H, 5.10; N, 7.47; Found: C, 58.02; H,
5.70; N, 7.50%: IR (cmꢁ1): 1645, m (
mC@N), 1475 and 1607, w

M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
113
Scheme 1. Structural representation of formation of Schiff base compounds (1–3).
(
m
C@C), 3565, br. (
m
OH), 1260, m (
m
NAC), 3470, m (
m
NAH stretch.),
1433 and 1596, w (
m
C@C), 3271, s (
m
OH), 1323, m (
m
NAC), 3485,
1563, w (
m
NAH bend.): 1H NMR (CDCl3, ppm): 1.37 (s, 9H, H2),
m ( NAH stretch.), 1577, w (m
m
NAH bend.): 1H NMR (CDCl3,
7.52–7.54 (d, 2H, H4, H40, 3J[1HA1H] = 8.4 Hz), 7.82–7.79 (d, 2H,
H5, H50, 3J[1HA1H] = 8.1 Hz), 8.44 (s, 1H, H6), 7.38 (s, 1H, H10),
ppm): 2.40 (s, 3H, H1), 7.20 (d, 2H, H3, H30, 3J[1HA1H] = 7.5 Hz),
7.90 (d, 2H, H4, H40, 3J[1HA1H] = 8.4 Hz), 8.0 (s, 1H, H6), 6.87–
6.90 (dd, 1H, H8), 6.85–6.80 (t, 1H, H9, 3J[1H-1H] = 15 Hz), 6.75–
6.78 (dd, H10), 10.57 (s, 1H, OH, NAHAO), 9.22 (s, 1H, NH), 3.81
(s, 3H, H13); 13C NMR (CDCl3, ppm): 21.6 (1C, C1), 144.8 (1C, C2),
130.1 (2C, C3, C30), 127.9 (2C, C4, C40), 134.5 (1C, C5), 147.3 (1C,
C6), 117.5 (1C, C7), 122.8 (1C, C8), 119.6 (1C, C9), 113.8 (1C,
C10), 119 (1C, C11), 151.5 (1C, C12), 56.0 (1C, C13); Molar Conduc-
7.35–7.42
(dd,
1H,
H10),
6.91–6.94
(d,
1H,
H13,
3J[1HA1H] = 8.7 Hz), 11.12 (s, 1H, OH, NAHAO), 9.22 (s, 1H, NH);
13C NMR (CDCl3, ppm): 35.2 (1C, C1), 31.3 (3C, C2), 155.4 (1C,
C3), 125.8 (2C, C4, C40), 128 (2C, C5, C50), 130.4 (1C, C6), 163.3
(1C, C7), 146 (1C, C8), 121.8 (1C, C9), 131 (1C, C10), 110.9 (1C,
C11), 133.9 (1C, C12), 119.1 (1C, C13), 156.9 (1C, C14); Molar Con-
ductance in 70% ethanol (Km
,
l
S cm2 molꢁ1): 15 at 25 °C: Solubil-
tance in 70% ethanol (Km, l
S cm2 molꢁ1): 4.6 at 25 °C: Solubility:
ity: chloroform, toluene, ethanol, methanol and DMSO.
chloroform, toluene, ethanol, methanol and DMSO.
Synthesis of N0-(2-hydroxy-3-methoxybenzylidene)-4-
methylbenzenesulfonohydrazide (3)
DNA interaction study assay
Yield: 90%: m.p.: 100–102 °C: Mol. Wt.: 320.4: Anal. Calc. for
UV–Visible Spectroscopy
C
15H16N2O4S: C, 56.24; H, 5.03; N, 8.74; S, 10.01; Found: C,
SS-DNA (50 mg) was dissolved by stirring for overnight in dou-
ble deionized water (pH = 7.0) and kept at 4 °C. Doubly distilled
56.30; H, 5.10; N, 8.80; S, 10.12%: IR (cmꢁ1): 1607, m (
mC@N),

114
M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
water was used to prepare buffers (20 mM Phosphate buffer
(NaH2PO4–Na2HPO4), pH = 7.2). A solution of (SS-DNA) in the buf-
fer gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) of
1.8, indicating that the DNA was sufficiently free of protein [18].
The DNA concentration was determined via absorption spectros-
copy using the molar absorption coefficient of 6600 Mꢁ1 cmꢁ1
(260 nm) for SS-DNA [19,20] and was found to be 1.4 ꢂ 10ꢁ4 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).
loaded with 66.6
(12 mg/mL in DMSO) to make 200
Tubes were then allowed to solidify in slanting position at room
temperature. Each tube was inoculated with 4 mm diameter piece
of inoculum from seven days old fungal culture. The media supple-
l
L
of compound from the stock solution
g/mL final concentration.
l
mented with DMSO and Turbinafine (200 lg/mL) were used as neg-
ative and positive control, respectively. The tubes were incubated
at 28 °C for 7 days and growth was determined by measuring lin-
ear growth (mm) and growth inhibition was calculated with refer-
ence to the negative control as shown in equation:
ꢀ
ꢁ
Linear growth of test sampleðmmÞ
Linear growth in ControlðmmÞ
%Growth inhibition ¼ 100ꢁ
ꢂ100
Enzymatic activity
Phosphatases are enzymes that catalyze the hydrolysis of esters
of phosphoric acid. They occur in the cells and extracellular fluids
of a wide range of organisms. This large and complex group of en-
zymes falls into four general types based on the chemical nature of
the substrate or the type of hydrolytic reaction that is catalyzed.
One group, the phosphomonoesterases, hydrolyzes monoesters of
Viscometry
Viscosity experiments were carried out using an Ubbelodhe vis-
cometer at a room temperature (25 1 °C). Flow time was mea-
sured with a digital stopwatch and each sample was measured
three times, and an average flow time was calculated. Data were
1/3
presented as (
where
pounds 1–3, and
g
/
go
)
vs. binding ratio (r) of [Compound]/[DNA],
phosphoric acid such as
a-lycerophosphate or glucose 6-phos-
g
is the relative viscosity of DNA in the presence of com-
phate. Some phosphomonoesterases are highly substrate-specific.
For example, in gluconeogenesis, fructose-1,6-bisphosphatase spe-
cifically converts fructose 1,6-bisphosphate to fructose 6-phos-
phate and inorganic phosphate. Other phosphomonoesterases
react with a broad range of substrates, which share common struc-
tural motifs. The phosphomonoesterases that lack substrate speci-
ficity are classified as acid or alkaline phosphatases based on their
pH optima. Acid phosphatases function best at around pH 5.0 and
are inhibited by fluoride ion but not by divalent cation-chelating
agents. The alkaline phosphatases have pH optima of about 9.0
and are not generally sensitive to fluoride ion but are inhibited
by divalent cation-chelating agents like EDTA [26]. Phosphatase
activity was assayed spectrophotometrically by using p-nitro-
phenyl phosphate (p-NPP) as a substrate. The continuous produc-
tion of p-nitrophenol (p-NP) was determined at 25 °C by
measuring absorbance at 405 nm in a reaction mixture containing
ALP from human serum in 0.1 M Na2CO3–NaHCO3 (sodium carbon-
ate–bicarbonate) buffer (pH = 10.1) [27]. The enzyme catalyzes the
hydrolysis of phosphate monoesters resulting in the formation of
inorganic phosphate and an alcohol. The identity of the alcohol
varies depending on the specific phosphatase. Alkaline phospha-
tase hydrolyzes the colorless, synthetic substrate p-nitrophenyl
phosphate to produce a yellow-coloured product, p-nitrophenol
and inorganic phosphate [26] as shown in Scheme 2.
g
o is the relative viscosity of DNA alone. Viscosity
values were calculated from the observed flow time of DNA-con-
taining solutions corrected for the flow time (to) of 20 mM phos-
phate buffer solution (pH 7.2) alone. The viscosity for DNA in the
presence and absence of the compound was calculated from the
following equations [21–23]:
g
¼ t ꢁ to
and
t ꢁ to
g
¼
to
Antibacterial assay
The synthesized compounds were tested against six bacterial
strains; two Gram-positive [Micrococcus luteus and Staphylococcus
aureus] and four Gram-negative [Escherichia coli, Pseudomonas
aeruginosa, Bordetella bronchiseptica and Klebsiella pneumonia].
The agar well-diffusion method was used for the determination
of antibacterial activity [24,25]. Broth culture (0.75 mL) containing
ca. 106 colony forming units (CFU) per millilitre of the test strain
was added to 75 mL of nutrient agar medium at 45 °C, mixed well,
and then poured into a 14 cm sterile petri plate. The media was al-
lowed to solidify, and 8 mm wells were dug with a sterile metallic
Stock solution of 20 lM inhibitor (HL) in one mL DMSO was
prepared at room temperature. The buffer and substrate were
mixed in 1:4 ratios to make the reagent solution. Then from the re-
borer. Then a DMSO solution of test sample (100 lL) at 1 mg/mL
was added to the respective wells. DMSO served as negative con-
trol, and the standard antibacterial drugs Roxyithromycin (1 mg/
mL) and Cefixime (1 mg/mL) were used as positive control. Tripli-
cate plates of each bacterial strain were prepared which were incu-
bated aerobically at 37 °C for 24 h. The activity was determined by
measuring the diameter of zone showing complete inhibition
(mm).
agent solution 2000
lL (2 mL) was taken in the cell and to which
40 L enzyme and varying concentrations of the inhibitor were
l
added. The spectrum of the alkaline phosphatase in the presence
and absence of inhibitor was measured using UV–Visible Spectro-
photometer. The release of yellow coloured p-nitrophenol chromo-
phore was monitored at 405 nm wavelength. Enzyme activity has
been expressed as the
lM of p-nitrophenol released per min for
5 min for various concentrations of the inhibitor. The inhibition
of enzyme by inhibitor was calculated by the following formula
[28]:
Antifungal assay
Antifungal activity against five fungal strains [Fusarium monili-
formis, Aspergillus niger, Fusarium solani, Mucor species and Aspergil-
lus fumigatus] was determined by using agar tube dilution method
[24,25]. Screw caped test tubes containing sabouraud dextrose
agar (SDA) medium (4 mL) were autoclaved at 121 °C for 15 min.
Tubes were allowed to cool at 50 °C and non solidified SDA was
D
nm=minuteꢂVmlðreagentþenzymeþinhibitorÞꢂD:F
18:5ꢂVmLof enzymetaken
Units=mLenzyme¼
ꢀ
D
0 min
A405nm = A405nm at time t min (e.g., 5 min) – A405nm at time

M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
115
Scheme 2. Hydrolysis of p-nitrophenyl phosphate catalyzed by alkaline phosphatase.
ꢀ D.F = dilution factor
mL was added. After 10 min the decrease in absorption was mea-
sured 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 re-
sults obtained were averaged [18].
ꢀ 18.5 = Millimolar extinction coefficient of p-nitrophenol at
405 nm
By the addition of increasing amounts of inhibitor the activity of
the enzyme decreased and at higher concentration it was almost
completely inhibited as shown in Fig. 9.
Cytotoxicity assay
Results and discussion
Cytotoxicity was studied by the brine-shrimp lethality assay
method [24,25]. Brine-shrimp (Artemia salina) eggs were hatched
in artificial sea water (3.8 g sea salt/L) at room temperature (22–
29 °C). After two days these shrimps were transferred to vials con-
taining 5 mL of artificial sea water (8 shrimps per vial) with 30, 50,
The Schiff base compounds (1–3) were prepared as described in
the experimental part, crystallized, dried and subjected to FT-IR, 1H
and 13C NMR techniques, single crystal X-ray techniques and ele-
mental analyses. The results of elemental analyses (C, H, N)
obtained are in good agreement with those calculated for the
suggested formula. The sharp melting point indicates the purity
of the synthesized compounds. The structures of compounds
(1–3) along with numbering are given in Scheme 2. Compounds
1 and 2 were also confirmed by single crystal analysis.
80 and 100 lg/mL in DMSO. After 24 h number of surviving
shrimps was counted. Data were analyzed with a slide write pro-
gram. The data represents the mean of three different experiments
ꢃ
done in duplicate. P < 0.05.
DPPH scavenging assay
FT-IR
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/
In the IR spectra of compounds 1–3 a sharp band observed at
3271 cmꢁ1 (1), 3565 cmꢁ1 (2) and 3271 cmꢁ1 (3) is assigned to free
OAH. A strong band attributable to C@N is observed at 1609 cmꢁ1
(1), 1645 cmꢁ1 (2) and 1607 cmꢁ1 (3). The stretching vibration of
medium intensity for NAH band occurs at 3480 cmꢁ1 (1),
Fig. 1. Ortep diagram along with numbering scheme of compound 1.

116
M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
Fig. 2. Ortep diagram along with numbering scheme of compound 2.
3470 cmꢁ1 (2) and 3485 cmꢁ1 (3) while a weak intensity peak for
bending vibration of N-H is observed at 1437 cmꢁ1 (1),
1563 cmꢁ1 (2) and 1577 cmꢁ1 (3). A peak of medium intensity as-
signed to the stretching vibration of NAC band occurs at
1297 cmꢁ1 (1), 1260 cmꢁ1 (2) and 1323 cmꢁ1 (3). The detail of IR
result is given in experimental section.
compounds 1–3 gives a broad singlet at 10.80, 11.12, 10.57 ppm,
respectively. This downfield shifting of OH proton is due to strong
intramolecular OAHAN hydrogen bonding, to which the chemical
shift of the proton is very sensitive [18]. A singlet is observed for
the NAH proton in compounds 1–3 at 9.80, 9.22 and 9.22 ppm,
respectively. In the 13C NMR spectra of compounds 1–3 the peak
at 147.7, 146 and 147.3 ppm, respectively belongs to the azome-
thine carbon (C@N). The remaining peaks are described in the
same positions as calculated by incremental methods [29].
NMR
The NMR spectra of compounds 1–3 were recorded in chloro-
form (CDCl3). The chemical shifts of the different types of protons
and carbons are given in experimental part. The formation of com-
pounds 1–3 was supported by the appearance of a sharp singlet
corresponding to the azomethine proton (AN@CHA) at 8.72,
8.44, 8.0 ppm, respectively. The hydroxyl proton in all the three
Crystal structures
The ORTEP diagrams along with numbering scheme of 4-tert-
butyl-N0-(2-hydroxy-3-methoxybenzylidene)benzohydrazide (1)
and N0-(5-bromo-2-hydroxybenzylidene)-4-tert-butylbenzohyd-
razide (2) are shown in Figs. 1 and 3 while their crystal data, se-
lected bond distances and angles are given in Tables 1 and 2,
respectively. Unit cells and H-bonds of compounds 1 and 3 are
shown in Figs. 2 and 4, respectively. The space groups of the crys-
tals are Pccn and P21/c, respectively. The bond lengths within the
phenyl ring range from 1.372(4) to 1.398(3) Å for (1) and
1.364(4) to 1.407(4) Å for (2), that are typical of aromatic character
[30]. Compound 1 exists as dimer while compound 2 as monomer.
The structure of the compound 2 consists of 1D-polymeric chain by
Table 1
Crystal data and structure refinement parameters for compounds 1 and 2.
Compound
Compound 2
1
Formula
Formula weight
Space group
Colour
C19H22N2O3
326.39
Pccn
C18H21BrN2O2
375.26
P21/c
White
White
a (Å)
b (Å)
c (Å)
25.7378(7)
26.1271(6)
10.5989(2)
90.00
90.00
90.00
7127.3(3)
16
1.217
0.083
2784
18.7332(6)
6.9099(2)
14.1120(6)
90.00
101.057(2)
90.00
1792.81(11)
4
1.457
2.311
808
296(2)
0.71073
2.22–25.24
3247
3247, 1966,
0.0478
Table 2
Selected bond lengths (Å) and bond angles (°) for compounds 1 and 2.
a
(°)
Compound 1
Compound 2
b (°)
Bond lengths
c
(°)
V (Å3)
Z
O4
N3
C20
N1
N2
C1
C2
C6
C3
C4
O1
O2
C20
N4
C21
C7
C8
C6
C3
C5
C4
C5
C1
C2
1.350(3)
1.378(3)
1.398(3)
1.277(3)
1.360(3)
1.395(3)
1.373(3)
1.398(3)
1.388(3)
1.372(4)
1.356(3)
1.369(3)
Br1
O1
N1
N1
N2
C1
C1
C1
C3
C2
C5
C2
N2
C7
C8
C6
C7
C2
C4
C3
C10
C14
1.900(3)
1.356(4)
1.384(4)
1.283(4)
1.349(4)
1.391(5)
1.441(4)
1.407(4)
1.369(5)
1.374(5)
1.389(4)
1.364(4)
d (g cmꢁ3
)
l
(Mo K
a
) (mmꢁ1
)
F(000)
T (K)
Radiation (Å) (Mo K
hMin–Max (°)
Total reflections
296(2)
a
)
0.71073
1.11–25.25
6430
6430, 3957,
0.0327
3957
Tot., Uniq. Data, R (int)
C9
C13
Observed data [I > 0.0 sigma(I)]
Nref, Npar w = 1/
1966
3247, 227
6430, 443
Bond angles
O1
C2
O2
N1
N2
C20
O3
C27
C29
C31
[r
2(Fo)2+(0.0721P)2 + 2.4714P] (for
C1
C1
C2
C7
C8
C21
C8
C28
C30
C34
C2
C6
C1
C6
117.8(2)
119.6(2)
115.3(2)
120.8(2)
115.4(2)
119.2(2)
121.5(2)
122.0(2)
122.4(2)
112.4(2)
N2
C2
N1
C1
C7
C6
C3
C3
C5
C4
C11
C13
C13
C18
116.2(3)
117.8(3)
117.9(3)
119.6(3)
118.7(3)
118.4(2)
121.2(3)
115.8(3)
121.6(3)
108.4(3)
compound 1) and w = 1/
2(Fo)2+(0.0438P)2 + 0.5330P] (for
compound 2) where P = [(Fo)2 + 2(Fc)2]/3
R, wR2, S
[r
O1
C1
C2
C2
0.0490,
0.1268,
1.014
0.00, 0.00
ꢁ0.205,
0.380
0.0404,
0.0849, 1.001
C9
C3
C4
C22
N2
C29
C31
C36
Br1
C9
C11
C9
C5
C10
C12
C14
C15
Max. and Av. shift/error
0.00, 0.00
ꢁ0.468, 0.385
Min. and max. resd. dens. [e/Å3]
C12
Goodness-of-fit
1.014
1.001

M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
117
Fig. 3. Absorption spectra of 1 mM of compound (1) in the absence (a) and presence of 5–80
lM (b–p) DNA. The arrow direction indicates increasing concentrations of DNA.
Inside graph is the plot of Ao/(A ꢁ Ao) vs. 1/[DNA] for the determination of binding constant and Gibb’s free energy of compound (1) – DNA adduct.
Fig. 4. Absorption spectra of 1 mM of compound (2) in the absence (a) and presence of 5–30
lM (b–g) DNA. The arrow direction indicates increasing concentrations of DNA.
Inside graph is the plot of Ao/(A ꢁ Ao) vs. 1/[DNA] for the determination of binding constant and Gibb’s free energy of compound (2) – DNA adduct.
H-bonding. The hydroxyl H atom is involved in an intramolecular
interaction with the nitrogen of the hydrazine moiety.
ent concentrations of DNA, respectively. It was observed that com-
pounds 1 and 3 have one strong absorption peak at 299.80 nm and
290 nm, respectively, with a shoulder peak at 330 nm. While com-
pound 2 shows four peaks at k1 = 337.20 nm, k2 = 300.40 nm,
k3 = 289 nm, k4 = 245.60 nm with a shoulder peak at 392.60 nm.
After interaction with increasing amount of DNA, all the peaks de-
creased gradually and the wavelengths have minor red shift of
1 nm for compound 1, 2 nm for compound 2 and 16 nm for com-
DNA interaction study by UV–Visible Spectroscopy
Electronic absorption spectra were initially used to examine the
interaction between compounds (1–3) and SS-DNA. Figs. 5–7 show
the UV–Visible spectra of compounds (1–3) interaction with differ-

118
M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
Fig. 5. Absorption spectra of 1 mM of compound (3) in the absence (a) and presence of 5–90
lM (b-p) DNA. The arrow direction indicates increasing concentrations of DNA.
Inside graph is the plot of Ao/(A ꢁ Ao) vs. 1/[DNA] for the determination of binding constant and Gibb’s free energy of compound (1) – DNA adduct.
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 [11]. Therefore, based on
this viewpoint, the interaction between compounds (1–3) and
SS-DNA could be noncovalent intercalative binding. After interca-
lating the base pairs of DNA, the pꢃ orbital of the intercalated li-
gand could couple with
the
pꢃ transition energy, and further resulting in the bathochro-
mism. On the other hand, the coupling of a orbital with partially
filled electrons decreases the transition probabilities hence results
hypochromic shift. Since hypochromism due to
pꢃ stacking
p orbital of base pairs, thus decreasing
p–
p
p–
interactions may appear in the case of the intercalative binding
mode, while bathochromism (red-shift) may be observed when
the DNA duplex is stabilized [31,32].
Based upon the variation in absorbance, the intrinsic binding
constant of the compound with DNA were determined according
to Benesi–Hildebrand equation [33]:
Fig. 6. Effects of increasing amount of Compounds (1–3) on relative viscosity of SS-
DNA at 25 0.1 °C. [DNA] = 7.2 M, r = 0, 6.9, 13.9, 20.8, 27.8.
l
A0
eG
ꢁ
eG
eG eHꢁG
1
¼
þ
ꢂ
A ꢁ A0 eHꢁG
ꢁ eG K½DNAꢄ
where K is the association/binding constant, A0 and A are the absor-
bances of the compound and its complex with DNA, respectively,
and eG and eH–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 (
DG) was deter-
mined from the equation:
D
G ¼ ꢁRTlnK
Fig. 7. % Inhibition of the ALP enzyme vs. concentration (
(1–3).
lM) of the Compound
where R is general gas constant (8.314 JKꢁ1 molꢁ1) and T is the tem-
perature (298 K).
The binding constants were found to be 2.9 ꢂ 104 Mꢁ1
,
pound 3, respectively. Long and Barton [10] had pointed out that
the absorption peaks shift of the small molecules after they inter-
acted with DNA could be as the clues to judge the binding mode
1.67 ꢂ 104 Mꢁ1 and 6.98 ꢂ 104 Mꢁ1, respectively while the Gibb’s
free energies were ꢁ11.08 kJ molꢁ1
ꢁ12 kJ molꢁ1, respectively.
,
ꢁ10.46 kJ molꢁ1 and

M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
119
Table 3
Antibacterial activity of compounds 1–3.
Compound
Average zone of inhibition (mm)
Staphylococcus aureus
Klebsiella pneumoniae
Micrococcus luteus
Pseudomonas aeruginosa
Bordetella bronchiseptica
Escherichia coli
1
2
18
16
18
17
20
22
16
15
19
15
20
19
3
20
20
25
20
22
20
Roxythromycin
Cefixime
36
22
26
21
35
30
20
16
35
30
26
22
MIC (lg/mL)
500
250
1.95
62.5
500
250
Concentration: 1 mg/mL of DMSO. Reference drugs, Roxythromycin and Cefixime 1 mg/mL.
Table 4
Antifungal activity of compounds 1–3.
Compound
Mean value of percent growth inhibition SD
Aspergillus Flavus
Fusarium solani
Aspergillus niger
70 0. 7
60
77 0.3
100
Mucor species
Aspergillus fumigates
1
2
3
70 0.5
70 1.6
71 0.4
55 0.7
75 0.8
100
80 1.5
80 0.3
85 0.5
100
65 0.9
60 1.3
80 1.2
100
1
80
1
Terbinafine
100
In vitro agar tube dilution method, concentration: 200
growth in vehicle control (mm).
lg/mL of DMSO. % Inhibition of fungal growth = 100 – gt/gc ꢂ 100. Gt = linear growth in test (mm) and gc = linear
DNA interaction study by viscometry
to good activity against five fungus strains. Since Gram-negative
To confirm the DNA binding modes, viscosity studies were car-
ried out. Hydrodynamic measurements that are sensitive to length
change (i.e., viscosity) are regarded as the least ambiguous and the
most critical tests of the binding model in solution. A classical
intercalation model resulted in the lengthening of the DNA helix
as the base pairs were separated to accommodate the binding com-
plex, leading to an increase in DNA viscosity. In contrast, a partial,
non-classical intercalation could bend (or kink) the DNA helix and
reduce its effective length and, concomitantly, its viscosity. A sig-
nificant increase in the viscosity of DNA on addition of compound
results due to the intercalation which leads to the separation
among the DNA base pairs to the increase in the effective size in
bacteria are considered a quantitative microbiological method for
testing beneficial and important drugs in both clinical and experi-
mental tumor chemotherapy [34], therefore the synthesized com-
pounds can be used for such purposes.
Though the exact biochemical mechanism is not completely
understood, the mode of action of antimicrobials may involve var-
ious targets in the microorganisms. The Schiff base compounds dis-
turb the respiration process of the cell and thus block the synthesis
of proteins, which restricts further growth of the organisms. This
enhancement in inhibiting the growth of bacteria and fungi can
also be explained on the basis of their structure. The azomethine
linkage in the synthesized complexes exhibit extensive biological
activities due to increased liposolubility of the molecules in cross-
ing cell membrane of the microorganism. The presence of electron
donor group (AOCH3) in compounds 1 and 3 also play a role in
enhancing their inhibition activity [35,36].
DNA which could be the reason for the increase in the viscosity
1/3
[18,33]. Plot of (
g/go
)
vs. [Compound]/[DNA] gives a measure
of the viscosity changes (Fig. 6). An increase in the relative viscos-
ity was observed on addition of the compounds 1–3 to DNA solu-
tion suggesting mainly intercalating binding nature of the
compounds.
Enzymatic activity
The compounds 1–3 were also screened for ALP inhibition
which revealed their strong affinity as inhibitor of the activity of
the enzyme. The enzymatic inhibition by compounds 1–3 is shown
in Fig. 7.
Antibacterial activity
The antibacterial activity data of the compounds 1–3 are given
in Table 3. Criteria for activity is based on inhibition zone (mm),
i.e., inhibition zone more than 20 mm shows significant activity,
for 18–20 mm inhibition activity is good, 15–17 mm is low, and
below 11–14 mm is non-significant [23,24]. The values obtained
for activity of compound 3 against all the six falls in the category
of significant activity while that for 1 and 2 fall in the category of
significant to good activity.
Cytotoxicity
All the synthesized compounds showed cytotoxic effect while
using brine shrimp as a substrate (See Supplementary data:
Figs. S1–S3). Among the tested compounds we found that com-
pound 3 is highly effective against brine shrimp. Furthermore, con-
centration of 100 lg/mL showed 100% death.
Antifungal activity
The synthesized compounds 1–3 were also tested for their anti-
fungal activity by agar tube diffusion method. The data are given in
Table 4. Criteria for activity is based on percent growth inhibition;
more than 70% growth inhibition shows significant activity, 60–
70% inhibition activity is good, 50–60% inhibition, activity is mod-
erate and below 50% inhibition activity is non-significant [23,24].
The investigation shows that the compounds 1–3 have significant
DPPH scavenging activity
The model of the scavenging of the stable DPPH radical is exten-
sively used to evaluate antioxidant activities in less time than
other methods. The reducing abilities of the compounds (1–3)
were determined by their interaction with the free stable radical
1,1-diphenyl-2-picryl-hydrazyl (DPPH) at different concentrations.

120
M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
130
Compound 2
A. Acid
104
78
52
26
0
10
20
30
50
100
150
Scheme 3. The proposed mechanism of antioxidant activity.
Fig. 9. Percent scavenging activity of DPPH vs. concentration (
(2).
lg/mL) of Compound
The proposed mechanism of antioxidant activity is shown in
Scheme 3. Antioxidants can react with DPPH and produce 1,1-di-
phenyl-2-picryl-hydrazine. Due to its odd electron DPPH gives a
strong absorption band at 517 nm appearing a deep violet colour
[37–39]. 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 elec-
trons 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 [37]. The
DPPH radical is a stable free radical (due to extensive delocaliza-
tion of the unpaired electron) having kmax at 517 nm. When this
compound abstracts a hydrogen radical from compounds (1–3)
then absorption vanishes due to the absence of free electron delo-
calization [39]. The percent scavenging activity of compounds (1–
3) was calculated as:
A0 ꢁ As
Fig. 10. Percent scavenging activity of DPPH vs. concentration
Compound (3).
(lg/mL) of
Scavenging activityð%Þ ¼
ꢂ 100
A0
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) [40].
compounds were as potent as ascorbic acid. Furthermore, com-
pound 3 was found more potent than ascorbic acid while using
both of the same concentration (Fig. 10).
The effects of the compounds (1–3) have been shown in Figs. 8–
10 with comparison to standard antioxidant ascorbic acid. The
plots show that with increase in concentration of the test samples
the intensity of radicals vanishes rapidly at a particular wavelength
(517 nm). The compounds (1–3) has scavenging activity due to the
OH group which can react with DPPH radical by the typical H-
abstraction reaction (HAT) to form a stable macromolecular radical
[41]. From all tested compounds we found that they have the capa-
bility of scavenging free radicals while using DPPH. All these
Conclusions
In summary, three Schiff bases of N0-substituted
benzohydrazide and sulfonohydrazide derivatives: 4-tert-butyl-
N0-(2-hydroxy-3-methoxybenzylidene)benzohydrazide (1), N0-
(5-bromo-2-hydroxybenzylidene)-4-tert-butylbenzohydrazide
(2) and N0-(2-hydroxy-3-methoxybenzylidene)-4-methylbenzene-
sulfonohydrazide (3) were synthesized by a simple condensation
reaction of primary amines and aldehydes. All these new com-
pounds were confirmed by elemental analysis, FT-IR, 1H, 13C
NMR spectroscopy and single crystal analysis. The in vitro
antibacterial and antifungal evaluation showed that all the
synthesized N0-substituted benzohydrazide derivatives exhibited
good to significant antimicrobial activities. The MIC value for anti-
bacterial activity ranges from 1.95 to 500 lg/mL. All the three com-
pounds interact with DNA via intercalation mode of interaction in
which the compound inserts itself into the base pair of DNA. This
binding mode of interaction is conformed by UV–Visible spectro-
scopic and viscometric techniques. They also act as a potent inhib-
itor of alkaline phosphatase enzyme. The synthesized compounds
were found an effective scavenging of the stable DPPH radical
DPPH as potent as standard antioxidant agent, ascorbic acid. They
also exhibit cytotoxic activity against brine shripm. Among the
studied three compounds, the biological activity in each filled is
Fig. 8. Percent scavenging activity of DPPH vs. concentration (
(1).
lg/mL) of Compound

M. Sirajuddin et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 116 (2013) 111–121
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Crystallographic data for the structures reported in this paper
have been deposited with the Cambridge Crystallographic Data
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of these information may be obtained free of charge from The
Director, CCDC, 12, Union Road, Cambridge CB2 1EZ [Fax: +44
(1223)336 033] or e-mail deposit@ccdc.cam.ac.uk or http://
www.ccdc.cam.ac.uk. Supplementary data associated with this
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