Salen, reduced salen and N-alkylated salen type compounds: Spectral characterization, theoretical investigation and biological studies

Article abstract of DOI:10.1016/j.saa.2012.11.100

Salen [2,2′-{propane-1,3-diylbis[nitrilo(E)methylylidene]}bis(6- methoxyphenol)], reduced salen [(2,2′-[propane-1,3-diylbis(iminomethylene) )]bis(6-methoxyphenol)] and N-alkylated salen [diethyl-2,2′-(propane-1,3- diylbis((2-hydroxy-3-methoxybenzyl) azanediyl))diacetate] compounds have been synthesized and characterized by IR, ¹H NMR, ¹³C NMR and UV-vis. spectroscopy. Molecular geometry of the title compounds in the ground state has been optimized by density functional method (B3LYP) with 6-31G basis set. Vibrational frequencies of the compounds were computed and compared with the experimental values. Tautomeric stability study of salen inferred that the enolimine form is more stable than its ketoenamine form in gas phase. The spectral behavior of salen in polar and nonpolar solvents was examined demonstrate the positive solvatochromism. The synthesized compounds have been studied with respect to their binding to calf thymus DNA showed that there were interactions between the compounds and DNA through a groove binding mode. Furthermore, the DNA cleavage activity of the compounds has been investigated by gel electrophoresis. The antioxidant properties of compounds were evaluated by DPPH method. The N-alkylated compound has a higher DPPH free radical scavenging activity. The antimicrobial activity was investigated on various gram positive and gram negative bacteria.

Full text of DOI:10.1016/j.saa.2012.11.100

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Salen, reduced salen and N-alkylated salen type compounds: Spectral
characterization, theoretical investigation and biological studies
⇑
P. Jeslin Kanaga Inba, B. Annaraj, S. Thalamuthu, M.A. Neelakantan
Chemistry Research Centre, National Engineering College, K.R. Nagar, Kovilpatti 628 503, Thoothukudi District, Tamil Nadu, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
Salen type compounds were synthesized and characterized. Molecular geometry of the compounds was
optimized by density functional method (B3LYP) with 6-31G basis set. The DNA binding value of the com-
pounds was determined. The antioxidant activity of the compounds was studied using DPPHÅ.
"
Salen type compounds were
synthesized and characterized.
Frontier molecular orbitals and
vibrational frequencies were
computed by DFT/B3LYP.
pH and solvent effects on UV–vis
spectra of the compounds were
studied.
"
"
"
"
DNA binding constants were
determined.
Antioxidant activities were
evaluated.
a r t i c l e i n f o
a b s t r a c t
Salen [2,20-{propane-1,3-diylbis[nitrilo(E)methylylidene]}bis(6-methoxyphenol)], reduced salen [(2,20-
[propane-1,3-diylbis(iminomethylene))]bis(6-methoxyphenol)] and N-alkylated salen [diethyl-2,20-(pro-
pane-1,3-diylbis((2-hydroxy-3-methoxybenzyl) azanediyl))diacetate] compounds have been synthesized
and characterized by IR, 1H NMR, 13C NMR and UV–vis. spectroscopy. Molecular geometry of the title
compounds in the ground state has been optimized by density functional method (B3LYP) with 6-31G
basis set. Vibrational frequencies of the compounds were computed and compared with the experimental
values. Tautomeric stability study of salen inferred that the enolimine form is more stable than its keto-
enamine form in gas phase. The spectral behavior of salen in polar and nonpolar solvents was examined
demonstrate the positive solvatochromism. The synthesized compounds have been studied with respect
to their binding to calf thymus DNA showed that there were interactions between the compounds and
DNA through a groove binding mode. Furthermore, the DNA cleavage activity of the compounds has been
investigated by gel electrophoresis. The antioxidant properties of compounds were evaluated by DPPH
method. The N-alkylated compound has a higher DPPH free radical scavenging activity. The antimicrobial
activity was investigated on various gram positive and gram negative bacteria.
Article history:
Received 2 November 2012
Received in revised form 19 November 2012
Accepted 27 November 2012
Available online 5 December 2012
Keywords:
Salen type compounds
Tautomerism
Solvent effects
DNA binding properties
Antioxidant activities
Ó 2012 Elsevier B.V. All rights reserved.
Introduction
and liphophilic properties [1]. Salen type compounds are used as
starting materials in the synthesis of antibiotics, antiallergic, anti-
The salen type compounds prepared by the condensation of o-
hydroxyaldehydes and diamine present versatile, steric, electronic
phlogistic and antitumor drugs [2–4]. A prototropic tautomeric
attitude has been recognized in 2-hydroxy Schiff bases are of inter-
est mainly due to the existence of OHꢀ ꢀ ꢀN and NHꢀ ꢀ ꢀO type hydro-
gen bonds. Hydrogen bonding interactions play roles in
preferential solvation and have been investigated because it is
present in large variety of chemical, biochemical and pharmacolog-
⇑
Corresponding author. Tel.: +91 9442505839; fax: +91 4632232749.
E-mail addresses: maneels@rediffmail.com, drmaneelakantan@gmail.com (M.A.
Neelakantan).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.11.100

P. Jeslin Kanaga Inba et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
301
ical events. In salen compounds, the imine nitrogen can acts as
intramolecular hydrogen bond acceptor and the phenolic oxygen
derivatives can act as intermolecular hydrogen bond acceptor.
The instability of salen type compounds can be overcome by reduc-
tion process. When these compounds are reduced at the imine
function, a more flexible, structurally related and basic salen deriv-
atives are obtained [5–7]. N-alkylation of reduced salen gives ester
derivatives. Schiff bases containing ester groups show remarkable
activity as plant growth hormone [8].
Binding of small molecules to DNA has been studied extensively
[9–12]. Small molecules can interact with DNA through non-cova-
lent modes namely, intercalation, groove binding and external
electrostatic binding [13–16]. To design effective chemotherapeu-
tic agents and better anticancer drugs, it is essential to explore the
interactions of small molecules with DNA. Free radicals play an
important role in the oxidative damage of biological systems
[17]. They can attack biological molecules such as lipids, proteins,
enzymes, DNA and RNA, leading to cell or tissue injury. Therefore
estimation of antioxidant activities of the synthesized compounds
is important [18].
Despite extensive work has been on salen ligands, little atten-
tion has been paid to the reduced salen and N-alkylated salen com-
pounds [19–22]. Hence, in continuation of our earlier works on
Schiff bases [23–25], the present investigation reports on the syn-
thesis and spectral characterization of salen, reduced salen and N-
alkylated salen compounds. Theoretical investigations of the syn-
thesized compounds were carried out using DFT/B3LYP/6-31G
method. This paper also describes the solvatochromism of the
synthesized salen in different solvents in the UV–visible spectra.
Comparative studies of the interactions of the synthesized com-
pounds with DNA as well as their antioxidatant activities were
investigated systematically. The experimental results suggest that
the synthesized compounds would have potential applications for
developing efficient antioxidants.
chloroform (25 mL). A brownish yellow colored solution was ob-
tained. The resulting reaction mixture was stirred well at room
temperature for 5 h and evaporated under vacuum to remove the
solvent. The solution was kept in a refrigerator for 2 days when
yellow colored solid precipitate of salen (L1) obtained. Then it
was filtered, washed with cold ethanol several times and dried
and finally preserved in a desiccator as a yellow solid. Yield: 88%;
m.p. 135 °C; Anal. calcd. for C19H22N2O4: C, 66.65; H, 6.48; N,
8.18%; Found: C, 66.28; H, 6.29; N, 8.01%. IR (KBr), cmꢁ1: 3448,
3290, 2945, 1631, 1467, 1384, 1271, 1082. 1H NMR (300 MHz,
CDCl3): dH = 13.9 (2H, s, OH), 8.3 (2H, s, CH@N), 6.7–6.9 (6H, m,
ArAH), 3.9 (6H, s, AOCH3), 3.7 (4H, s, NACH2), 2.1 (2H, s, CH2).
kmax: 221, 265, 339, 430 nm.
Synthesis of 2,20-[propane-1,3-diylbis(iminomethylene)]bis(6-
methoxyphenol) (L2)
Approximately 2 g (5 mmol) of the salen (L1) was dissolved in
30 mL methanol and sodium borohydride was added drop wise un-
til the formation of a colorless solution. The resulting solution was
stirred well at room temperature. After 5 h, the solution was ex-
tracted with chloroform and the extracted organic layer was
washed with saturated ammonium chloride solution, dried over
anhydrous Na2SO4, evaporated under vacuum to remove the sol-
vent and kept on the bench for 24 h. The white colored precipitate
formed at the end of this period was the reduced salen (L2). Yield:
80%; m.p. 107 °C; Anal. Calcd. for C19H26N2O4: C, 65.87; H, 7.56; N,
8.09%; Found: C, 65.52; H, 7.38; N, 7.98%. IR (KBr), cmꢁ1: 3419,
3230, 3323 1271, 1236, 1107, 1084. 1H NMR (300 MHz, CDCl3):
dH = 6.6–6.8 (6H, m, ArAH), 4.1 (4H, s, CH2ANH), 3.8 (6H, s,
AOCH3), 2.7 (4H, s, NHACH2), 2.2 (2H, s, ANH), 1.8 (2H, s, ACH2).
kmax: 275, 345 nm.
Synthesis of diethyl-2,20-(propane-1,3-diylbis((2-hydroxy-3-
methoxybenzyl) azanediyl))diacetate (L3)
Approximately 2 g (5 mmol) of the reduced salen (L2) was dis-
solved in 20 mL acetonitrile by mixing and potassium carbonate
(4.1 g, 30 mmol) was added. The mixture was stirred well under
nitrogen atmosphere. Then ethylbromoacetate (3.19 mL, 25 mmol)
was added drop wise and stirring was continued for 24 h. The
resulting solution was extracted and washed with chloroform
and water. The organic layer was separated and dried using anhy-
drous Na2SO4, filtered and evaporated in vacuum. The filtrate was
allowed to stand overnight in air when a semisolid mass appeared
at the bottom. The solvent was evaporated off to give the ester (L3)
as pale brown oil. The product obtained was purified by column
chromatography and is obtained as yellow oil. Yield: 60%; Anal.
Calcd. for C27H38N2O8: C, 62.53; H, 7.38; N, 5.40%; Found: C,
Experimental
Materials and methods
All chemicals employed for the synthesis were of analytical re-
agent grade and of highest purity available. o-vanillin and 1,3-
diaminopropane were purchased from Sigma Aldrich and used as
received. Solvents used for the studies were purified and dried by
standard procedures. CT DNA and pUC19 DNA were purchased from
Genei, Bangalore and used without purification. Tris HCl and ethi-
dium bromide were obtained from HiMedia. Tris HClANaCl buffer
solution was prepared with double distilled water. 2,2-diphenyl-
1-picryl-hydrazyl (DPPH) was purchased from Aldrich.
Microanalytical data were obtained on a Thermo Finnigan Flash
EA 1112 series CHN analyzer for C, H and N. The infrared (FT-IR)
spectra were recorded on a FTIR Shimadzu 8400S spectrophotom-
eter in the region 4000–400 cmꢁ1. The 1H and 13C NMR of the com-
pounds in CDCl3 were recorded on Bruker AV 300 MHz. The
electronic spectra were recorded from 200 to 500 nm using Shima-
dzu UV-2450 spectrophotometer at room temperature.
CHO
N
N
CHCl3
5 h
H2N
NH2
OH
HO
OH
O
O
O
L1
CH3OH NaBH4
Synthesis of salen (L1), reduced salen (L2) and N-alkylated salen (L3)
compounds
O
O
O
K2CO3
BrCH2COOC2H5
O
N
H
OH
N
H
HO
Synthesis of the title compounds is shown in Scheme 1.
N
OH
N
HO
O
O
Synthesis of 2,20-{propane-1,3-diylbis[nitrilo(E)methylylidene]}bis(6-
methoxy phenol) (L1)
o-vanillin (20 mmol, 3.04 g) dissolved in chloroform (25 mL)
was mixed with 1,3-diaminopropane (10 mmol, 0.833 mL) in
O
O
L2
Scheme 1. Synthesis of the salen type compounds (L1–L3).
L3

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Table 1
The experimental and computed vibrational frequencies of salen (L1), reduced salen (L2) and N-alkylated salen (L3) compounds (cmꢁ1).
Assignments
L1
L2
L3
Experimental
DFT/ B3LYP
Experimental
DFT/ B3LYP
Experimental
DFT/B3LYP
OAH str
3448
3290
2945,2839
1631
3775
3346
3271,3243
1832
3419
3279
2928
–
3589
3236
3104
–
3441
3293
2933
–
3464
3229
3000
–
CAH (aromatic) stretching
CAH (CH2) stretching
C@N stretching
CAN stretching
NAH stretching
C@C (aromatic) stretching
C@O (ester)
CAO (phenolic) stretching
CAOAC sym. stretching
CAOAC asym. stretching
OAH bend (in plane)
OAH bend (out of plane)
CAH(aromatic) bend (in plane)
CAH(aromatic) bend (out of plane)
–
–
1467
–
–
1728
–
1582
1340
1107
3323,3230
1479
–
1271
1076
1236
1384,1352
729
1299
3478
1520
–
1423
1175
1389
1492
912
1193
–
1292
–
1479
1747
1280
1084
1273
1379
754
1527
1714
1368
1189
1347
1478
863
–
1271
1082
1255
1534
1384,1359
742
1170
881, 837
1492,1444
926
1382
1077,974
1186
833
1230
1008
1184
860
1264
1029
Fig. 1. 1H NMR spectrum of N-alkylated salen (L3) in CDCl3.
62.35; H, 7.25; N, 5.28%. IR (KBr), cmꢁ1: 3441, 3293, 1747, 1280,
1273, 1193, 1084. 1H NMR (300 MHz, CDCl3): dH = 6.5–6.8 (6H,
m, ArAH), 4.2 (4H, s, CH2CH3), 3.8 (6H, s, OCH3), 3.6 (4H, s, CH2AN),
3.4 (4H, s, ANCH2COO), 2.6 (4H, s, ANCH2CH2), 1.8 (2H, s, ANCH2-
CH2), 1.2 (6H, s, ACH2). 13C NMR (300 MHz, CDCl3): dC = 174.12
(COO), 156.51 (CAOH), 151.41 (CAOCH3), 61.85 (OCH2CH3), 59.12
(NCH2COO), 54.20 (CH2N), 45.37 (NCH2CH2), 23.04 (OCH2CH3).
kmax: 267, 333 nm.
(DFT) method with Becke0s three-parameters hybrid functional
using Lee–Yang–Par correlation functional (B3LYP) employing 6-
31G basis set as implemented in Gaussian 03W [26]. In addition,
vibrational frequencies were computed at the corresponding opti-
mized geometry using the same theory level. The vibrational fre-
quencies calculations showed no imaginary frequencies that
ascertained the optimized structure were stable. Total energy
and HOMO and LUMO energies for the title compounds (L1–L3)
were obtained at B3LYP/6-31G level.
Computational details
DNA binding measurements
All computations were performed using Gaussian 03W program
running under Windows XP. Full geometry optimization of the title
compounds was performed by using Density Functional Theory
The DNA binding experiments were performed at room temper-
ature. Buffer solution 5 mM Tris HCl and 50 mM NaCl were used for

P. Jeslin Kanaga Inba et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
303
Fig. 2. 13C NMR spectrum of N-alkylated salen (L3) in CDCl3.
½DNAꢂ
½DNAꢂ
1
¼
þ
ð1Þ
N
N
ðea
ꢁ
ef
Þ
ð
eb
ꢁ
ef
Þ
Kbðeb
ꢁ
ef
Þ
N
H
N
H
H
H
O
O
O
O
O
O
O
O
where ea
,
ef and eb are the apparent, free and bound compound
½DNAꢂ
extinction coefficients respectively. In the plots of
versus
ð
ꢁef Þ
[DNA], Kb is given by the ratio of slope to the intercepte.a
Enol form
Keto form
Scheme 2. Keto-enol tautomerism of salen, L1.
DNA cleavage experiment
the absorption titrations. A solution of CT DNA in the buffer gave a
ratio of UV absorbance at 260 and 280 nm of about 1.8–1.9:1, indi-
cating that the CT DNA was sufficiently free from protein [27]. The
concentration of DNA was measured using its extinction coefficient
at 260 nm (6600 mol Lꢁ1 cmꢁ1) [28]. Stock solutions were stored at
4 °C and used within 4 days. Concentrated stock solutions of the
compounds were prepared by dissolving the compounds in DMSO
and diluting suitably with the buffer to the required concentrations
for all the experiments. The absorption titrations of the compounds
The DNA cleavage experiment was conducted using pUC 19
DNA by gel electrophoresis with the corresponding compound in
the presence of H2O2 as oxidant. The reaction mixture was pre-
pared as follows: 10
DMSO, 1 L of H2O2 followed by dilution with buffer 50 mM
Tris–HCl and 50 mM NaCl to a total volume of 25 L. The samples
lL of pUC 19 DNA, 5 lL of the compound in
l
l
were incubated at 37 °C for 1 h in the presence and absence of the
compound. It is then loaded on 1% agarose gel after mixing with
3 lL of loading dye (0.25% bromophenol and 40% sucrose). The
samples were electrophoresed at 100 V using Tris-boric acid-EDTA
buffer (pH = 8.0) until the bromophenol blue reached to one third
of the gel. The gel was stained using ethidium bromide for
10 min and the bands were visualized and photographed under a
UV Trans illuminator.
in buffer were performed using a fixed concentration (20 lM) to
which increments of the DNA stock solution were added
½DNAꢂ
(R ¼
¼ 0; 2; 4; 6; 8and10). Compound–DNA solutions were
½Compoundꢂ
allowed to incubate for 30 min before the absorption spectra were
recorded. From the absorption data, the intrinsic binding constant
Kb was determined using the equation [29]

304
P. Jeslin Kanaga Inba et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
Antimicrobial activity
(a)
y = 1.0054x + 217.27
3500
2500
1500
500
The ligands L1, L2 and L3 were tested for their in vitro antibac-
R2 = 0.9922
terial activity against Streptococcus pyogenes and Staphylococus as
Gram positive bacteria and Escherichia Coli, Klebsiella, Aeromonas
and Serratia as Gram negative bacteria by well diffusion method
[33] using Ampicillin and Amoxicillin as standards. The liquid
medium containing the bacterial subcultures was autoclaved for
20 min at 121 °C and at 15 lb pressure before inoculation. The bac-
teria were then cultured for 24 h at 37 °C in an incubator. Mueller–
Hinton nutrient agar was poured over sterile 90 mm Petri dishes
and allowed to solidify. Wells were made on the agar medium
inoculated with microorganism. The test compounds in DMSO
solution were added into the well using suitable techniques and
the plates were incubated at 24 h at 37 °C. During this period,
the test solution diffused and the growth of the inoculated micro-
organism was affected. The inhibition zone was developed and
measured at the end of the incubation period. Experiments were
performed in triplicate and standard deviation was calculated.
500
1500
2500
3500
3500
3500
Experimental
(b)
(c)
3500
2500
1500
500
y = 0.9877x + 145.51
R2 = 0.9953
Results and discussion
500
1500
2500
All the synthesized compounds are stable at room temperature.
The compounds are soluble in most of the organic solvents and
insoluble in water. The analytical data of the compounds are given
in Section 2.
Experimental
3500
2500
1500
500
y = 0.9525x + 146.46
R2 = 0.9975
IR spectra
The FT-IR spectral data of the title compounds (L1–L3) are given
in Table 1 and Fig. S1. The IR spectrum of the salen, L1 exhibits a
broad band centered at 3448 cmꢁ1 is due to the formation of strong
intramolecular and intermolecular hydrogen bonding of phenolic
AOH group. A strong band at 1631 cmꢁ1 is due to the azomethine
group vibration. The CAO stretching vibration of the phenolic part
of o-vanillin is appeared at 1271 cmꢁ1. In the reduced salen (L2),
the spectrum exhibits a medium peak at 3230 cmꢁ1 is ascribed
to the NAH stretching vibration, confirming that the imine group
of the salen has been reduced. The absence of ACH@NA group in
L2 is further confirmed from the disappearance of the typical
strong band at 1631 cmꢁ1 due to imine vibration appears in L1.
The band appeared at 1107 cmꢁ1 is due to the CANH vibration of
the reduced part of the azomethine group. In the N-alkylated salen
(L3), the bands at 1193 and 1747 cmꢁ1 can be attributed to the
vibrations of the CAN and C@O of ester groups respectively. These
data confirms the formation of salen, reduced salen and N-alkyl-
ated salen compounds.
500
1500
2500
Experimental
Fig. 3. Correlation graphs of calculated and experimental IR frequencies of: (a) L1
(b) L2 and (c) L3.
Antioxidant property
Antioxidant activity of the synthesized compounds was ex-
pressed from their radical scavenging ability against the stable free
radical, 2,2-diphenyl-1-picrylhydrazyl (DPPHÅ). The DPPHÅ scav-
enging capacity was measured according to the following proce-
dure [30,31]. The concentration of DPPHÅ used for antioxidant
activity was 50 lM. Different concentrations of the synthesized
compounds in methanol was added to DPPHÅ in methanol solution.
The reaction tubes were wrapped in aluminum foil and shaken vig-
orously and kept at room temperature for 30 min in dark. The
reduction of the DPPHÅ was monitored by observing the decrease
in absorbance at 517 nm using a spectrophotometer. The radical
scavenging capacity of the antioxidants was expressed in terms
of % inhibition and IC50. The capability to scavenge the DPPHÅ
was calculated using the following equation [32].
1H and 13C NMR
The 1H and 13C NMR spectra of L1–L3 were recorded in CDCl3.
The signal at 8.3 d observed in L1 can be attributed to the azome-
thine proton demonstrated the formation of salen compound (L1).
The phenolic AOH proton of L1 shows signal at 13.9 d (Fig. S2). The
aromatic protons exhibit multiplets in the 6.7–6.9 range. The
methoxy protons show singlet at 3.9 d. The signals at 3.7 and 2.1
can be attributed to the ACH2 protons of 1,3-diaminopropane.
From these data, the formation of salen (L1) is confirmed. The dis-
appearance of azomethine proton signal at 8.3 and appearance of a
new signal at 4.1 (methylene proton of benzyl part) in the spec-
trum of L2 clearly demonstrates the reduction of azomethine group
(L2) (Fig. S3). Aromatic protons give signals in the region 6.6–6.8.
The peak at 3.8 is considered as methoxy protons. The signal at
ðAcontrol ꢁ Asample
Þ
% Inhibition ¼
ꢃ 100
ð2Þ
Acontrol
where Acontrol is the absorbance of DPPHÅ in methanol solution with-
out an antioxidant, and Asample is the absorbance of DPPHÅ in the
presence of an antioxidant. The IC50 value is the concentration of
the antioxidant required to scavenge 50% DPPHÅ and is calculated
from the inhibition curve.
2.7 d is assigned to
at 1.8 d is considered as b methylene protons of the amine part.
a methylene proton of amine part. The peak

P. Jeslin Kanaga Inba et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
305
LUMO+1 (-0.02735)
LUMO+1 (0.00882)
LUMO+1(-0.00678)
LUMO (-0.03005)
LUMO (0.00641)
LUMO (-0.0293)
HOMO (-0.18823)
HOMO (-0.17098)
HOMO (-0.20689)
HOMO-1 (-0.20896)
HOMO-1 (-0.2018)
HOMO-1 (-0.1955)
L1
L2
L3
Fig. 4. The frontier molecular orbital of L1, L2 and L3.
Table 2
is given in Fig. 2. In the spectrum, the signal at 156.5 d can be as-
signed to phenolic carbon atom. Aromatic ring carbon atoms of
L3 are in the 116–127 d range. The signals at 54.2 and 174.1 d
can be attributed to carbon attached to nitrogen atom and carbonyl
carbon of the ester group. The signals at 45.3, 61.8 and 23.4 d are
due to the carbon atoms of the CH2 group in 1,3-diaminopropane,
CH2 and CH3 groups in ester part respectively. The position of car-
bon atoms clearly confirms the formation of N-alkylated salen (L3).
The computed total energy, HOMO, LUMO energies and energy gap with DFT /(B3LYP)
6-31G basis set for L1–L3.
Parameters
(a.u.)
L1
L2
L3
Enol form
Keto form
Total
ꢁ1147.29060 ꢁ1147.5713
ꢁ1150.07599 ꢁ1762.49670
Energy
EHOMO
ELUMO
ꢁ0.20689
ꢁ0.03005
0.17684
ꢁ0.1829
ꢁ0.07026
0.11264
ꢁ0.17098
0.00641
0.17739
ꢁ0.18823
ꢁ0.0293
0.15893
ELUMO
–
EHOMO
Electronic absorption spectra
The electronic absorption spectra of the synthesized salen type
compounds are given in Fig. S4. Salen (L1) shows UV bands at 221,
These data support the formation of reduced salen (L2). In the 1H
NMR spectrum of L3, the signals at 4.0–4.2 and 1.2 are due to
the methylene and methyl protons of ester (Fig. 1). Multiplets of
aromatic protons appear in the region 6.5–6.8. The peak at 3.8 is
assigned as methoxy protons. The signals at 3.6, 3.3, 2.6 and 1.8
are assigned to methylene protons from phenyl ring, ANACH2-
265, 339 and 430 nm. The bands at 221 and 265 nm (
and 23400 Mꢁ1 cmꢁ1) are attributed to pꢄ transitions of the
aromatic ring, 339 nm (
max = 6700 Mꢁ1 cmꢁ1) is due to
max = 1500 -
emax = 56800
p
?
e
p ?
pꢄ
transition of the azomethine and the band at 430 nm (
e
Mꢁ1 cmꢁ1) is assigned to n ? pꢄ transition of azomethine group.
There is a shoulder at 290 nm, which may be due to the delocaliza-
tion of hydrogen from AOH groups to give keto and enol form
(Scheme 2) [34]. The reduced salen compound shows two bands
ACOO,
a to amino part and b to amino part respectively. These data
supports the formation of N-alkylated salen (L3). The labeling of
carbon atoms and the 13C NMR spectrum of N-alkylated compound

306
P. Jeslin Kanaga Inba et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
Fig. 5. UV–vis spectra of salen L1 (1 ꢃ 10ꢁ5 M) in solvents with different polarity
range.
Table 3
Electronic spectral data of salen (L1) in polar and nonpolar solvents.
Solvent
Dielectric
constant
Benzene
ring
p
Azomethine
Emax
ring
p
?
pꢄ
(kcal molꢁ1
)
?
pꢄ
1,4-Dioxane
Benzene
Chloroform
Ethyl acetate
2-Propanol
Methanol
2.21
2.28
4.81
6.0
18.3
32.6
37.5
254
279
261
264
263
264
261
272
329
332
330
332
331
338
328
335
86.90
86.12
86.64
86.12
86.37
84.59
87.17
85.35
Acetonitrile
Dimethylsulphoxide 47
Fig. 6. pH dependent UV–vis. spectra of L1 (a) pH = 1.52–10.02 and (b) pH = 4.96–
11.98.
at 275 nm and 345 nm. The high intensity band at 275 nm is attrib-
uted to
pꢄ transition of aromatic ring and low intensity band
at 345 nm is characteristic of
pꢄ transition due to H-bonding
p
?
p
?
induced changes of OH proton-donor aromatic molecules and
amine NH [35]. These bands are shifted in N-alkylated salen (L3)
and are observed at 267 and 333 nm.
Table 4
Electronic absorption spectral properties of L1–L3 with CT-DNA.
Compound
kmax (nm)
D
k (nm)
aH%
Kb (Mꢁ1
)
Computational studies
Free
Bound
L1
L2
L3
265.4
279.8
272.8
266.8
281.8
277.2
1.4
2
4.4
12.07
22.20
13.04
4 ꢃ 104
1.25 ꢃ 104
1.23 ꢃ 105
Optimized geometry
The structures of the title compounds have been optimized
using the B3LYP method at 6-31G level. The salen compound exits
in keto-enol tautomerism. The enolimine (OAHꢀ ꢀ ꢀN) and keto-
amine (NAHꢀ ꢀ ꢀO) tautomers for the compound L1 is shown in
Scheme 2. To investigate the tautomeric stability, optimization at
B3LYP/6-31G level for both enolimine and ketoamine forms of L1
was performed in gas phase. The value of the total energy calcu-
lated in gas phase for the enolimine form of L1 is ꢁ1147.29060
a.u., which is lower than the ketoamine form (ꢁ1147.5713 a.u.).
This clearly demonstrates that the enol imine form is more stable
than the ketoamine form in gas phase.
a
H% = [(Afree–Abound)/Afree] ꢃ 100%.
compared with the calculated results (Table 1). The assignment
of the experimental frequencies is based on the observed band fre-
quencies in the infrared spectra by establishing one to one correla-
tion between observed and theoretically calculated frequencies.
The calculated frequencies are higher than the observed values
for the majority of the normal modes. The reason for the discrep-
ancy between the experimental and computed spectra is that the
experimental value is an inharmonic frequency while the calcu-
lated value is a harmonic frequency. To make a comparison with
the experimental observation, correlation graphs (Fig. 3) were
drawn. From the correlation graphs, it is clear that the experimen-
tal fundamentals are found to have good correlation with the com-
puted values.
Vibrational assignments
Harmonic vibrational frequencies of the title compounds were
calculated using the DFT/B3LYP method with the 6-31G basis set
using the Gaussian 03W program package [26]. The vibrational
band assignments were made using the Gauss-View molecular
visualization program. The experimentally observed peaks were

P. Jeslin Kanaga Inba et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
307
Fig. 8. Trends in the inhibition of DPPH radicals by L1, L2 and L3 at different
concentrations.
Table 5
The antibacterial activities of L1–L3 by well diffusion method (zone formation in mm,
standard deviation 2).
Bacterial strains Diameter of inhibition zone of bacteria in different
compounds
L1
L2
L3
Ampicillin
Amoxicillin
S. pyogenes
Staphylococus
E. Coli
Klebsiella
Aeromonas
Serratia
13
14
11
12
8
5
6
10
R
5
6
5
8
R
R
7
8
16
10
12
14
12
10
14
10
14
16
10
10
14
R – Resistant.
implies an electron density transfer to the CAC bond of the ben-
zene ring from CH2ANHA group. The HOMO–LUMO energy gap
was calculated at DFT-B3LYP method using 6-31G basis set
(Table 2) reflects the chemical activity of the molecule. The LUMO
as an electron acceptor represents the ability to obtain an electron
and HOMO represents the ability to donate an electron. The smal-
ler the LUMO and HOMO energy gap computed in the present
investigation suggests that the HOMO electrons can be easily ex-
cited. Moreover, a lower HOMO–LUMO energy gap explains the
fact that eventual charge transfer interaction is taking place within
the molecule. The calculated physicochemical properties such as
total energies, HOMO and LUMO energies are given in Table 2.
Fig. 7. Absorption spectra of: (a) L1 (b) L2 and (c) L3 (20 lM) in 5 mM Tris–HCl
buffer at pH = 7.2, in the absence (R = 0) and presence (R = 2, 4, 6, 8 and 10) of
increasing amounts of CT DNA. Arrow shows the intensity changes upon increasing
DNA concentration. Inset: Plot of [DNA]/ea–ef versus [DNA].
Solvatochromaticity of the salen (L1) in different organic solvents
The salen (L1) shows band at 430 nm in methanol gives infor-
mation about ketoenol tautomerism of the compound (Fig. 5).
The UV–vis spectrum of L1 is taken in polar and non-polar solvents
in the 200–500 nm range and the spectral data are summarized in
Table 3. The absorption band at 335 nm (in DMSO) and 329 nm (in
Frontier molecular orbitals
The electronic absorption corresponds to the transition from
ground to the first excited state and is mainly described by one
electron excitation from the highest occupied molecular orbital
(HOMO) to the lowest unoccupied molecular orbital (LUMO) [36].
Fig. 4 shows the distributions and energy levels of the HOMO –
1, HOMO, LUMO, and LUMO – 1 orbitals computed at the B3LYP/
6-31G level for the title compounds. From the HOMO–LUMO orbi-
1,4-Dioxane) are attributed to
p ?
pꢄ transitions. However, a new
absorption band at 430 nm was observed in the spectrum of L1 in
polar protic solvents like methanol and 2-propanol which was not
present in the case of nonpolar and polar aprotic solvents. In non-
polar solvents, L1 is present predominantly in the enolimine form
as confirmed by NMR spectra taken in CDCl3. The signals of the hy-
droxyl and azomethine protons for L1 appear at 13.9 ppm and
tal picture, the filled
p orbital (HOMO) is mostly located in be-
tween the aromatic ring and the unfilled pꢄ orbital (LUMO) on
the aromatic ring. Consequently, the HOMO ? LUMO transition

308
P. Jeslin Kanaga Inba et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 104 (2013) 300–309
8.3 ppm respectively (Fig. S2). Their shapes and positions are char-
acteristic of the enolimine form with a strong intramolecular
OHꢀ ꢀ ꢀN@C bond [37]. Thus, the absorption band at 328–338 nm
arises from the enolimine form and the band at 430 nm arises from
Nuclease activity
The ability of the synthesized compounds to perform DNA
cleavage is carried out using pUC19 DNA by agarose gel electro-
phoresis (Fig. S5). The tests were performed under aerobic condi-
the ketoamine form in solution. The Emax values of
p
?
pꢄ transi-
tions at the compound concentration of 40 lM. The cleavage of
tion of the salen in different solvents were calculated by the fol-
lowing equation [38]:
pUC19 DNA induced by L1, L2 and L3 results in the conversion of
supercoiled form (SC) to nicked circular (NC) form. The nuclease
activity is measured from the amount of form I (SC) diminish
and partly converted to form II (NC) and follows the order
L3 > L1 > L2. The variation of DNA cleavage efficiency of the synthe-
sized compounds may be due to the different binding affinity of the
compound to DNA. The compound with a greater DNA binding pro-
pensity shows better nuclease activity.
Emaxðkcal molꢁ1Þ ¼ 28591=kmaxðnmÞ
ð3Þ
To find the sign, the value of
DE was calculated from the Emax of
the most nonpolar solvent and the most polar solvent.
D
E ¼ Emax of the most nonpolar solvent
ꢁ Emax of the most polar solvent
Antioxidant activity
The antioxidant activity of synthesized compounds was mea-
sured in terms of their hydrogen donating or radical scavenging
capability against stable radical DPPH by UV–vis. spectrophotome-
ter. The antioxidants containing phenols (ArOH) react with DPPHÅ
via two different mechanisms: (i) a direct donation of phenol H-
atom and (ii) an electron transfer process from ArOH or its phenox-
ide anion (ArOꢁ) to DPPHÅ [41]. The pathway of reaction depends
upon the nature of the solvent or the redox potentials of the spe-
cies involved. The experiment was carried out in methanol capable
of forming strong hydrogen bonds with the ArOH molecules and
the electron transfer mechanism becomes important. The decrease
of DPPHÅ absorbance with addition of compounds results in the
scavenging of the radical by electron transfer process from ArOH
or its phenoxide anion (ArOꢁ) to DPPHÅ [42]
The salen shows a red shift and positive solvatochromism when
the polarity of the solvent is increased. This confirms that the mol-
ecule is more polarized in the excited state (i.e.) the molecule is
stabilized by the polar solvents in the excited state.
UV–vis. spectra of salen (L1) in acidic and basic solutions
The spectrophotometric titration of salen in methanol was car-
ried out in acidic and basic medium to explain the deprotonation
process. The spectrum of salen (L1) gives three absorptions in the
range 200–500 nm as
a function of pH (1.52–11.98). From
pH = 1.52 to 7.52, the three peak positions (220, 264 and
341 nm) remain invariant suggests that L1 is in protonated form
in this pH range (Fig. 6a). By increasing the pH to 9.15, the absorp-
tion at 220 nm is shifted to 239 nm (bathochromic shift), and the
other two peaks at 264 and 341 nm start to decrease sharply in
intensity and a new peak occurs at 282 and 394 nm. These peaks
at 282 and 394 nm grows in intensity up to pH = 11.98 with com-
plete disappearance of the original peaks at 264 and 341 nm
(Fig. 6b). These results indicate that L1 is in deprotonated form
with pH > 9.15. There are two isosbestic points for the series of
curves from pH = 1.52 to 11.98 at 245 nm and 280 nm. At lower
pH, L1 is in the protonated form whereas at higher pH L1 is in
the deprotonated form.
ArOH þ DPPHÅ ! DPPHꢁ þ ArOHÅþ ! DPPHH þ ArOÅ
All the synthesized compounds show DPPHÅ radical scavenging
capability due to the presence of o-methoxy substitution which sta-
bilizes the aryloxyl radical [43]. The radical scavenging capacity of
the compounds calculated by Eq. (2) and is expressed in terms of
IC50 (50% inhibitory concentration). The IC50 values of L1, L2 and
L3 are 1022, 259.8 and 96.4 lM respectively (Fig. 8). Lower value
indicates the higher radical scavenging capacity. This implies that
the free radical scavenging activity of N-alkylated salen compound
is greater than the salen and reduced salen compounds
(L1 < L2 < L3). The lower IC50 value observed for N-alkylated salen
demonstrates that the compound has a potential to be applied as
scavenger to eliminate the free radicals. However, the IC50 values
of synthesized compounds are much higher than the positive con-
Biological activities
DNA binding studies
The UV–vis. absorption spectra of the compounds (L1–L3) in
buffer solution show intense absorption bands at 265, 280, and
273 nm, respectively (Table 4). These absorption bands are per-
turbed by the addition of increasing amounts of DNA (Fig. 7). Addi-
tion of CT DNA to the test compounds induce hyperchromic
responses of about 12.07% at 265 nm for L1, 22.2% at 280 nm for
L2 and 13.04% at 273 nm for L3 at R = 10. The hyperchromism
accompanied by a small shift in kmax is consistent with groove
binding leading to small perturbations [39]. In order to compare
quantitatively, the binding constants (Kb) were calculated using
trol like ascorbic acid, 11.55 lM [44].
Antimicrobial activity
The invitro antibacterial activity of the title compounds was
tested against six human pathogenic microorganisms, viz., S. pyog-
enes and Staphylococus as gram positive bacteria and E. Coli, Klebsi-
ella, Aeromonas and Serratia as gram negative bacteria by well
diffusion method using ampicillin and amoxicillin as standards
(Table 5). The results of antimicrobial studies of L1, L2 and L3 are
given in Fig. S6. A close examination of the results showed that
the salen compound, L1 is effective against all the bacteria tested.
Also, L1 shows higher activity for Staphylococus and Serratia than
the control. The reduced salen, L2 does not show any activity
against Klebsiella, but has moderate activity against other microor-
ganisms. The N-alkylated salen, L3 does not show any activity
against E. Coli and Klebsiella and has moderate activity against
other organisms. The remarkable activity of the salen may be due
to the presence of the azomethine and hydroxyl groups in the
molecule.
Eq. (1). The value of Kb determined from the ratio of slope to inter-
½DNAꢂ
cept from the plot of ð
Þ versus [DNA] is 4 ꢃ 104, 1.25 ꢃ 104 and
1.23 ꢃ 105 Mꢁ1 for L1,eLaꢁ2efand L3 respectively. The magnitude of the
binding constant values clearly showed that all the compounds
bound with CT DNA and the binding strength increases in the order
L2 < L1 < L3 (Table 4). These results suggest that molecular features
containing N-alkyl group on the side chain are associated with
higher affinity binding to DNA. However, the Kb values obtained
here are lower than the value reported for the classical intercalator,
ethidium bromide [40].

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In this work, a salen, reduced salen and N-alkylated salen have
been synthesized and characterized. The salen in solvent media is
found to exist in keto and enol forms. DFT study of salen inferred
that the enolimine form is more stable than its ketoamine form
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