Optical properties and fluorescence quenching of biologically active ethyl 4-(4-: N, N -dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1 H -indeno[1,2- b] pyridine-3-carboxylate (DDPC) dye as a probe to determine CMC of surfactants

Article abstract of DOI:10.1039/c6ra02814d

4-(4-N,N-Dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) was prepared via the multi-component reaction of indane-1,3-dione with 4-(dimethylamino)benzaldehyde, ethyl acetoacetate and ammonium acetate. Data obtained from FT-IR, ¹H-NMR, ¹³C-NMR, EI-MS and elemental analysis were consistent with the chemical structure of the newly prepared DDPC. Electronic absorption and emission spectra of DDPC were measured in different solvents. The DDPC dye exhibits a red shift in its emission spectrum as the solvent polarity increases, indicating a large change in the dipole moment upon excitation due to intramolecular charge transfer within the excited DDPC molecules. Excited state intermolecular hydrogen bonding has an effect upon the energy of the emission spectrum and the fluorescence quantum yield of the DDPC molecules. The DDPC dye undergoes solubilization into different micelles and can be used as a probe to determine the critical micelle concentration (CMC) of SDS and CTAB. The DDPC dye can also be used to probe of the polarity and hydrogen bonding properties of its local micro-environment. The antibacterial activity of DDPC was tested in vitro for the first time by using a disk diffusion assay against two Gram-positive and two Gram-negative bacteria, and the minimum inhibitory concentration (MIC) was also determined with reference to the standard drug tetracycline.

Full text of DOI:10.1039/c6ra02814d

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: S. A. Khan, A. M.
Asiri and S. H. Al-Thaqafy1, RSC Adv., 2016, DOI: 10.1039/C6RA02814D.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
Optical properties and fluorescence quenching of biological active
ethyl 4-(4-N,N-dimethylamino phenyl)-2-methyl-5-oxo-4,5-dihydro-
1H-indeno[1,2-b]pyridine-3-carboxylate (DDPC) dye as probe to
determine CMC of surfactants
Salman A. Khan1,*, Abdullah M. Asiri1,2, Saad H. AlꢀThaqafy1,
1Chemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box
80203, Jeddah 21589, Saudi Arabia
2Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz
University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
*Author to whom correspondence should be addressed; eꢀmail:
sahmad_phd@yahoo.co.in.
Abstract
indeno[1,2ꢀb]pyridineꢀ3ꢀcarboxylate (DDPC) was prepared by multi component
reaction of indaneꢀ1,3ꢀdione with 4ꢀ(dimethylamino)benzaldehyde, ethyl
4ꢀ(4ꢀN,Nꢀdimethylaminoꢀphenyl)ꢀ2ꢀmethylꢀ5ꢀoxoꢀ4,5ꢀdihydroꢀ1Hꢀ
acetoacetate and ammonium acetate. Data obtained from FTꢀIR, 1HꢀNMR, 13CꢀNMR,
EIꢀMS and elemental analysis were consistent with chemical structure of newly
prepared DDPC. Electronic absorption and emission spectrum of DDPC have been
measured in different solvents. DDPC dye exhibits red shift in emission spectrum as
solvent polarity increases, indicating a large change in dipole moment of DDPC
molecule upon excitation due to intramolecular charge transfer in excited DDPC.
Excited state intermolecular hydrogen bonding affect on the energy of emission
spectrum and fluorescence quantum yield of DDPC molecule. DDPC dye undergoes
solubilization in different micelles and may be used as a probe to determine the
critical micelle concentration (CMC) of SDS and CTAB. DDPC dye can also use as
probe of the polarity and hydrogen bonding properties of its local microenvironment.
The antiꢀbacterial activity of the DDPC was first tested in vitro by the disk diffusion
assay against two Gramꢀpositive and two Gramꢀnegative bacteria, and then the
1

RSC Advances
Page 2 of 28
View Article Online
DOI: 10.1039/C6RA02814D
minimum inhibitory concentration (MIC) was determined with the reference of
standard drug Tetracycline.
Keywords: DDPC, Stokes shift, Oscillator strength, Dipole moment, fluorescence
quantum yield, Micellization
2

Page 3 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
Introduction
Heterocyclic compound with the especial references of pyridine occupy special place
and have attracted considerable attention because of their broad pharmacological
4
activities, including antiꢀbacterial, antiꢀtumor, anticancer,1ꢀ3 antiviral,
antiꢀ
5
6
7
8
inflammatory, antimicrobial, antiꢀdiabetic,
antihypertensive and osteogenic
9
activities, in addition to treatment of CNS disorders. 10 Various synthetic methods
have been reported for the synthesis of pyridine derivative, biꢀcyclic heterocyclic
compounds such as pyrazoloꢀpyridine, thiazoloꢀpyridine, triazoloꢀpyridine have
synthesized by the cyclization of pyridine. 11 It's also used as ligands for the metal
complexes by the coordinate with the transition metal complexes. 12 Due to presence
of long π bond conjugation system in pyridine derivative its also used in the felids of
martial science such as nonlinear optical properties, 13 photonic materials, 14 devices,
16
17
optical limiting,15 electrochemical sensing,
lightꢀemitting devices,
langmuir
19
film, 18 and solar cell materials.
However, the pyridine has been used extensively
for the photoꢀalignment and photoꢀcrosslinking unit in polymers. Physicochemical
characteristics, such as, solvatochromic, piezochromic, oscillator strength, dipole
moment, fluorescence quantum yield and photostability, are also most important
studies for determining the physical behavior of compounds. 19 Donor (D) ꢀπ ꢀacceptor
(A) long π bond conjugate system gives the good physicochemical behavior due to the
20
intramolecular charge transfer by πꢀbond from the donor group to acceptor group.
On the basis of literature survey we find that lot of work have been done on
chromophores,21 to the best of our knowledge their is not much deep investigation on
the prescient topic which we are reporting first time, therefore due to the possible
importance of the D ꢀπ ꢀA chromophores our interest in the development of
heterocycle ꢀbased D ꢀπ ꢀA chromophores. In the present study, we wish to report the
3

RSC Advances
Page 4 of 28
View Article Online
DOI: 10.1039/C6RA02814D
synthesis of new heterocycleꢀbased chromophore. Oneꢀpot multicomponent reactions
(MCR) have conventional significant attention in synthetic chemistry as they can
produce target products from readily available starting materials in one reaction step
without isolating the intermediates thus reducing reaction times, labor cost, and waste
production. 22 Therefore in the present paper we are reporting the synthesis of 4ꢀ(4ꢀ
N,Nꢀdimethylaminoꢀphenyl)ꢀ 2 ꢀ methyl ꢀ 5ꢀ oxo ꢀ 4, 5ꢀdihydroꢀ1Hꢀindeno [1,2ꢀb]
pyridineꢀ3 carboxylate (DDPC) by MCR and physicochemical studies such as
oscillator strength, dipole moment, fluorescence quantum yield, fluorescence
quenching in organized media. DDPC dye undergoes solubilization in different
micelles and used as a probe to determine the critical micelle concentration (CMC) of
SDS and CTAB .
Experimental
Apparatus
1
FTꢀIR spectra were recorded on a Nicolet Magna 520 FTꢀIR spectrometer. HꢀNMR
and 13CꢀNMR experiments were performed in CDCl3 on a Brucker DPX 600 MHz
spectrometer using tetramethyl silane (TMS) as internal standard at room temperature.
Melting points were recorded on a Thomas Hoover capillary melting apparatus
without correction. UVꢀVis electronic absorption spectra were acquired on a
Shimadzu UVꢀ1650 PC spectrophotometer. Absorption spectra were collected using a
1 cm quartz cell. Steady state fluorescence spectra were measured using Shimadzu RF
5301 PC spectrofluorophotometer with a rectangular quartz cell. Emission spectra
were monitored at right angle. All fluorescence spectra were blank subtracted before
proceeding in data analyses.
Chemicals and reagents
4

Page 5 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
The appropriate chemical indaneꢀ1,3ꢀdione, 4ꢀ(dimethylamino)benzaldehyde, ethyl
acetoacetate and ammonium acetate was purchased from Acros Organic. Other
reagents and solvents (A.R.) were obtained commercially and used without further
purification, except dimethylformamide (DMF), ethanol and methanol.
4-(4-N,N-dimethylamino
phenyl)-2-methyl-5-oxo-4,5-dihydro-1H-indeno[1,2-
b]pyridine-3-carboxylate (DDPC)
To an alcoholic solution (50 mL) of indaneꢀ1,3ꢀdione 2 (0.01 mol), 4ꢀ
(dimethylamino)benzaldehyde (0.01 mol), ethyl acetoacetate 3 (0.01 mol), ammonium
acetate (0.02 mol) and a drop of piperidine were added and the mixture was refluxed
for 2 h. The reaction mixture was concentrated to half of its original volume and
allowed to cool in an iceꢀchest. The solid thus separated was filtered, washed with ice
cold aqueous ethanol and crystallized from petroleum ether (60–80°C)ꢀchloroform (1:
1) (Scheme 1).
Dark Brown solid: m.p. 122°C; EIMS m/z (rel. int.%): 391 (72) [M + 1]+; IR (KBr)
νmax cm–1: 3254 (NꢀH), 2964 (CꢀH), 1656 (C=O), 1585 (C=C); 1246 (CꢀN); 1H NMR
(600 MXz CDCl3) (δ/ppm): 9.16 (s, NH), 8.51ꢀ 6.47 (m, 9H, CH aromatic) 4.07 (s,
3H, ꢀCH3), 4.00 (s, 3H, ꢀCH3), 3.96 (s, 3H, ꢀCH3), 3.85ꢀ3.75 (q, CH2ꢀCH3), 1.61 (t, ꢀ
CH2ꢀCH3); 13CNMR (CDCl3) δ: 168.22 (C=O), 156.25 (pyridine C), 139.91, 134.76,
134.61, 124.81, 122.86, 122.78 (ArꢀC). Anal.calc. for C24H24N2O3: C, 74.21, H, 6.23;
N, 7.21 Found: C, 74.18, H, 5.97, N, 6.16.
5

RSC Advances
Page 6 of 28
View Article Online
DOI: 10.1039/C6RA02814D
CHO
O
O
O
H3C
O
CH3
+
+
O
N
H3C
CH3
2h, EtOH
NH4OAC
Piperidine
H3C
CH3
N
H3C
O
O
O
H3C
N
H
(DDPC)
Scheme 1: Synthesis of DDPC
The fluorescence quantum yield (φf) of DDPC (1 x 10ꢀ5 M) was evaluated in different
solvents. Rhodamine 6G (1 x 10ꢀ5 M) in ethanol (φf = 0.94)) was selected as a
standard sample since rhodamine 6G absorbs using the same excitation wavelength
(λex = 385 nm) of DDPC in ethanol and same concentration of the solution DPPC and
23
rhodamine 6G) to obtain the absorption spectra.
It is, therefore, expected that the
same number of photons are absorbed by both samples (Rhodamine 6G and DDPC).
The fluorescence quantum yield of DDPC can be related to that of the standard by the
following relationship: 24
I x Ar x n2
Φ
= Φr
(1)
Ir x A x n2
r
6

Page 7 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
Where φ is the quantum yield, I is the integrated emission intensity, A is the
absorbance at excitation wavelength, and n is the refractive index of the solvent. The
subscript r refers to the reference fluorophore of known quantum yield.
Organism culture and in vitro screening
Antibacterial activity was 4ꢀ(4ꢀN,Nꢀdimethylamino phenyl)ꢀ2ꢀmethylꢀ5ꢀoxoꢀ
4,5ꢀdihydroꢀ1Hꢀindeno[1,2ꢀb]pyridineꢀ3ꢀcarboxylate (DDPC) done by the disk
diffusion method with minor modifications. S. aureus, S. pyogenes, S. typhimurium
and E. coli were subꢀcultured in BHI medium and incubated for 18 h at 37 ºC, and
then the bacterial cells were suspended, according to the McFarland protocol in saline
solution to produce a suspension of about 10ꢀ5 CFU mLꢀ1: 10 ꢁL of this suspension
was mixed with 10 mL of sterile antibiotic agar at 40 ºC and poured onto an agar plate
in a laminar flow cabinet. Five paper disks (6.0 mm diameter) were fixed onto
nutrient agar plate. 1mg of DDPC was dissolved in 100 ꢁL DMSO to prepare stock
solution to form different concentration 10, 20, 25, 50, and 100 ꢁg/ꢁL of DDPC
different concentration were poured over disk plate on to it. Tetracycline (30 ꢁg/disk)
was used as standard drug (positive control). DMSO poured disk was used as negative
control. The susceptibility of the bacteria to the DDPC was determined by the
formation of an inhibitory zone after 18 h of incubation at 36 ºC (Table 2) reports the
inhibition zones (mm) of DDPC and the controls. The minimum inhibitory
concentration (MIC) was evaluated by the macro dilution test using standard
inoculums of 10ꢀ5 CFL mLꢀ1. Serial dilutions of the DDPC, previously dissolved in
dimethyl sulfoxide (DMSO) were prepared to final concentrations of 512, 256, 128,
64, 32, 16, 8, 4, 2 and 1 ꢁg/ mL to each tube was added 100ꢁL of 24 h old inoculum.
The MIC defined as the lowest concentration of the DDPC which inhibits the visible
growth after 18 h was determined visually after incubation for 18 h at 37 ºC and the
7

RSC Advances
Page 8 of 28
View Article Online
DOI: 10.1039/C6RA02814D
results are presented in table 2. Tests using DMSO and tetracycline as negative and
positive controls were also performed.
Results and discussion
Chemistry
4ꢀ(4ꢀN,Nꢀdimethylaminoꢀphenyl)ꢀ2ꢀmethylꢀ5ꢀoxoꢀ4,5ꢀdihydroꢀ1Hꢀindeno[1,2ꢀ
b]pyridineꢀ3ꢀcarboxylate (DDPC) was prepared by multi component reaction of
indaneꢀ1,3ꢀdione
with 4ꢀ(dimethylamino)benzaldehyde,
ethyl acetoacetate,
25
1
ammonium acetate.
The purified dye was characterized by the FTꢀIR, HꢀNMR,
13CꢀNMR, EIꢀMS m/z (rel. int.%), and elemental analysis. FTꢀIR spectra of DDPC
show that a characteristic band at 1676 cmꢀ1 of the ν (C=O) peak for indaneꢀ1,3ꢀdione
that is shifted to a lower frequency of 1656 cmꢀ1 for DDPC. This is due to the one
C=O is utilized for the formation of pyridine. The IR spectrum of DDPC shows the
characteristic band at 3254 cmꢀ1 due to presence –NH group. IR spectra shows sharp
peak at 1246 cmꢀ1 due presence of CꢀNꢀC stretch which is conform to formation of
1
pyridine ring. The HꢀNMR spectra of DDPC measured at room temperature shows
one singlet at 9.16 ppm for the NH. The appearance of multiplets at δ 8.51ꢀ 6.47 was
due to aromatic protons and three singlet at δ 4.07, 4.00 and 3.96 for corresponding to
the three methyl group present in the DDPC. Moreover, 13C NMR (CDCl3) spectra of
DDPC was recorded in CDCl3 and spectral signals are in good agreement with the
probable structures. The carbonyl carbon of the DDPC usually appears at δ 168.22 in
it 13C NMR spectrum. 13CꢀNMR spectra showed signals in the range of δ 139.91ꢀ
122.78 ppm due to aryl carbon. Details of 13CꢀNMR spectra of DDPC are given in the
experimental section. Finally characteristic peaks were observed in the mass spectra
of DDPC. The mass spectrum of DDPC shows a molecular ion peak (M+.) m/z 391.
8

Page 9 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
Spectral behavior of DDPC in different media
UVꢀvis absorption spectra of the DDPC (1 x 10ꢀ5 M) was measured in various nonꢀ
polar, polar aprotic and polar protic solvents such as ethanol, methanol,
dimethylsulfoxide, dimethylformamide, chloroform, dichloromethane, carbon
tetrachloride, acetonitrile, dioxan, tetrahydrofuran. Fig 1 shows absorption spectra of
a1 x10ꢀ5 mol dmꢀ3 solution of DDPC in these solvents as a sample. As it can be seen
from Fig 1, in the all solvent tested the main band of DDPC, located in the spectral
range 371ꢀ400 nm. Shorter wavelength band in UV region observed for studies DDPC
in different solvent system is assigned to π to π* transition of the benzenoid system
toward the other ring which is characterized by high electron donation and electron
accepting character present in its structure. On excitation at 385 nm and the range of λ
of emission between 395 nm to 700 nm, the emission spectrum of DDPC (1 x 10ꢀ5 M)
shows smooth correlation with increasing polarities of the solvent, broad and red
shifted (Fig 2 & Table 1) as the solvent polarity increases. The redꢀshift from 475 nm
in CCl4 to 548 in DMSO indicates that photoinduced intramolecular charge transfer
(ICT) occurs in the singlet excited state and therefore the polarity of DDPC increases
26
on excitation.
The red shift of the fluorescence peak in alcoholic solvents are
assigned to solute – solvent hydrogen bonding interaction in the singlet excited state
which causes red shift in the observed spectra (Table 1). 27
A simplified description of hydrogen bonding of DDPC is shown in Scheme 2. Type
(a) hydrogen bonding is strengthened in the excited state, since the charge density at
the carbonyl oxygen is enhanced in the ICT excited state. On the other hand, type (b)
hydrogen bonding is weakened on photoexcitation, because the charge densities at the
Nꢀ(CH3)2 decrease in the excited state.
9

RSC Advances
Page 10 of 28
View Article Online
DOI: 10.1039/C6RA02814D
ROH
b
N
ROH
a
O
O
O
H3C
N
H
Scheme 2 : Type of hydrogen bonding of DDPC
The energy of absorption (Ea) and emission (Ef) spectra of the DDPC in different
solvents correlated with the empirical Dimroth polarity parameter ET (30) of the
solvent (Fig. 3). 28 A linear correlation between the energy of absorption and emission
versus polarity of solvents was obtained (Equation 2 and 3), implying potential
application of these parameters to probe the microenvironment of DDPC.
Ea = 75.17 – 0.1032 × ET (30)
Ef = 68.28 – 0.257 × ET (30)
(2)
(3)
Analysis of solvatochromic behavior allows estimating the difference in the dipole
moment between the excited and ground states (ꢀꢁeꢀ ꢀꢁg). This was achieved by
29
applying the simplified Lippert – Mataga equation (4, 5).
10

Page 11 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
2
2(ꢁe
−
ꢁg
)
ꢀ
ν st
=
ꢀf + Const.
(4)
(5)
hca3
k −1
n2 −1
2n2 +1
ꢀf
=
−
2k +1
Where ꢀυ is the Stokes shift which increases with increase in the solvent polarity to
pointing to stronger stabilization of singlet excited state in polar solvent, h is Planck
constant, c is the speed of light and a is the Onsager cavity radius, k and n are the
dielectric constant and refractive index of the solvent, respectively. The constant
represents higher order terms which are usually neglected. The Onsager cavity radius
30
was taken as 5.7 Å.
Fig 4 shows the plot of Stokes shift versus the orientation
polarizability (ꢀf). The changes of dipole moment (ꢀꢁ ) upon excitation was
calculated from slope of the plot and the cavity radius is ꢀꢁ = 5.58 Debye. This
change in dipole moment is caused by redistribution of atomic charges in the excited
state as a result of charge transfer from the electron rich –Nꢀ(CH3)2 group to electron
acceptor ketoꢀgroup fragment.
The oscillator strength ( f ) and transition dipole moment (ꢁ12) of electronic transition
for DFTP from ground to excited singlet state (So → S1) was calculated in different
31
solvents using the following equation (6, 7).
f
= ∫ ε (ν ) dν
4.32×10−9
(6)
(7)
f
2
ꢁ12
=
4.72 ×10−7 Emax
11

RSC Advances
Page 12 of 28
View Article Online
DOI: 10.1039/C6RA02814D
Where ε the numerical value for molar decadic extinction coefficient is measured in
dm3 molꢀ1cmꢀ1 and υ is the value of wavenumber measured in cmꢀ1 and Emax is the
energy maximum of absorption band in cmꢀ1. The values of ƒ and (ꢁ12) are listed in
Table 1 and indicates that the So → S1 is strongly allowed transition
The empirical Dimroth polarity parameter, ET (30) and EN of DDPC was also
T
calculated according to the following equation. 32, 33
ET (solvent) − 30.7
ETN
=
(8)
(9)
32.4
28591
λmax
ET (solvent) =
where λmax corresponds to the peak wavelength (nm) in the red region of the
intramolecular charge transfer absorption of DDPC. The red (bathochromic) shift
from CCl4 to DMSO indicates that photoinduced intramolecular charge transfer (ICT)
occurs in the singlet excited state, and the polarity of DDPC, therefore, increases on
excitation.
Fluorescence quantum yield
The fluorescence quantum yield (φf) of DDPC depends strongly on the solvent
properties (Table 1). The fluorescence quantum yield can be correlated with ET(30) of
the solvent, where ET(30) is the solvent polarity parameter introduced by Reichardt.34
The fluorescence quantum yield of DDPC increases with increasing solvent polarity
from 0.16 in a nonꢀpolar solvent CCl4 to 0.43 in a moderately polar solvent dioxan;
with a further increase in solvent polarity the fluorescence quantum yield seems to
decrease, i.e., 0.29 in a strongly polar solvent, DMSO. This indicates the occurrence
of negative solvatokinetic effect and positive solvatokinetic effect during the course
12

Page 13 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
35
of increasing solvent polarity.
One main reason for the negative solvatokinetic
effect (increase φf with a suitable enhancement of ICT) could be due to the
biradicaloid charge transfer involving the unꢀbridged double bonds and the other
cause could be related to the proximity effect for compounds with nꢀл and лꢀл*
electron configuration. In other words, in nonꢀpolar solvents, these effects will result
in effective nonradiative decay of the excited states. In strong polar solvents, the
fluorescence quantum yield decreases, due to large degree of intramolecular charge
transfer, which causes an increase in the rate of radiationless relaxation of an excited
state, giving rise to positive solvatokinetic effect (reduction in φf by strong ICT).
Moreover, the much lower fluorescence quantum yields in proton solvents can be
attributed to the hydrogen bond interaction between the molecule and surrounding
solvent, which results in an additional nonradiative decay as observed in other dipolar
molecules.
Effect of surfactant on emission spectrum of DDPC
The emission spectrum of 1 x 10ꢀ5 mol dmꢀ3 of DDPC has also measured in cationic
micelle cetyltrimethyl ammonium bromide (CTAB) and anionic micelle sodium
dodecyl sulphate (SDS). As shown in Fig 6 and Fig 7, the emission intensity of DDPC
increases with increasing the concentration of surfactant, an abrupt change in
fluorescence intensity is observed at surfactant concentration of 7.30 x10ꢀ4and 7.45 x
10ꢀ3 mol dmꢀ3, We have used Carpena’s method 36 to obtain the value of cmc from the
data of emission intensity and found the same cmc value shown in Fig. 6 and Fig. 7 as
swhich are very close to the critical micelle concentration of CTAB and SDS, 37 thus
DDPC can be employed as a probe to determine the CMC of a surfactants. It was well
known that aromatic molecules were generally solubilized in the palisade layer of
38, 39
micelle.
Thus the enhancement of emission intensity is attributed to the passage
13

RSC Advances
Page 14 of 28
View Article Online
DOI: 10.1039/C6RA02814D
of dye molecule from the aqueous bulk solution to the palisade layer of micelle. The
decrease in polarity of the microenvironment around dye molecule results in the
reduction of nonꢀradiative rate from ICT state to lowꢀlaying singlet or triplet state due
to the enlargement the energy gap between them, which leads to an increase in
emission intensity. Critical micelle concentration (CMC) of SDS and CTAB with
DDPC were further conformed by the conductometric method.
Fluorescence quenching of DDPC with alcoholic solvents
The fluorescence quenching of DDPC in dioxan (On excitation at 385 nm) (
λ
=
ex
385 nm) was studied by using different concentration of some polar protic solvents of
different acidity, such as methanol, ethanol, 2ꢀpropanol and butanol as quenchers
Figure 8(a)ꢀ11 a). As follows from these figures, the fluorescence spectra undergo
very complex changes on adding different concentration of alcoholic solvents , i.e.,
they are shifted to longer wavelength, possess changed half widths and band profiles
of the emission spectrum. This behavior indicates, that in such a solution an extra
factor contributes to the well known dipoleꢀdipole interaction, i.e., hydrogenꢀbonding
40
interactions between the DDPC with the alcohol.
The Stern–Volmer constants
(KSV) were calculated from the Stern– Volmer plots shown in Fig 8(b)ꢀ 11(b). The
KSV constant was determined as 0.206, 0.169, 0.99 and 0.094 Mꢀ1 in methanol,
ethanol, 2ꢀpropanol and butanol respectively, which increases according to the acidity
(α) of the alcohol. It seems that the KSV value in case of methanol is higher than that
in the other solvents, which indicates that the possibility of hydrogen bonding with
solute increases with the decreeing the number of carbon atoms in the solvent
molecule. The dependence of fluorescence characteristics on solvent properties imply
a potential application of DDPC to probe of the polarity and hydrogen bonding
properties of it local microenvironment.
14

Page 15 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
I0 / I f =1 + Ksv [Q]
(10)
where Io and If are the relative integrated fluorescence intensities without and with the
quencher concentration [Q] and Ksv (SternꢀVolmer constant).
Antibacterial activity
Disc-diffusion assay
4ꢀ(4ꢀN,Nꢀdimethylaminoꢀphenyl)ꢀ2ꢀmethylꢀ5ꢀoxoꢀ4,5ꢀdihydroꢀ1Hꢀindeno[1,2ꢀ
b]pyridineꢀ3ꢀcarboxylate (DDPC) was tested for their antibacterial activities by discꢀ
diffusion method using nutrient broth medium [contained (g/L): beef extract 3 g;
41
peptone 5 g; pH 7.0.
The Gramꢀpositive bacteria and Gramꢀnegative bacteria
utilized in this study are S. aureus, S. pyogenes, S. typhimurium and E. coli. In the
discꢀdiffusion method, sterile paper discs (0.5 mm) impregnated with DDPC
dissolved in dimethylsulfoxide (DMSO) at concentration 100 ꢁg/mL were used.
Then, the paper discs impregnated with the solution of the test DDPC were placed on
the surface of the media inoculated with the microorganism. The plates were
ο
incubated at 35 C for 24 h. After incubation, the growth inhibition zones are shown
in table 2.
Assessment of minimum inhibitory concentration (MIC)
The MIC of synthesized DDPC and standard drug were investigated against two gram
positive and two gram negative bacteria using broth dilution method (BDM). The data
is reported as MIC which is defined as the lowest concentration required to inhibit
90% growth in comparison to control (absence of DDPC) for each isolate. Table 2
summarizes the in vitro susceptibilities of both the types of isolates against DDPC.
Evaluation of MIC showed that DDPC active in vitro against all the tested
15

RSC Advances
Page 16 of 28
View Article Online
DOI: 10.1039/C6RA02814D
microorganisms with varying degrees of inhibition, within the reference range.
Conclusion
Novel donor, accepter heterocyclic chromophore (4ꢀ(4ꢀN,Nꢀdimethylaminoꢀphenyl)ꢀ
2ꢀmethylꢀ5ꢀoxoꢀ4,5ꢀdihydroꢀ1 ꢀindeno[1,2 ]pyridineꢀ3ꢀcarboxylate) (DDPC) was
reaction of indaneꢀ1,3ꢀdione with 4ꢀ
ethyl acetoacetate, ammonium acetate and
H
b
prepared by multi component
(dimethylamino)benzaldehyde,
characterized by various spectral techniques. Optical properties of DDPC dye
including singlet absorption, extinction coefficient, stokes shift, oscillator strength,
dipole moment and fluorescence quantum yield were investigated on the basis of the
polarity of solvent. The absorption and emission spectra of DDPC exhibit an
intramolecular charge transfer band; which showed a positive solavotochromism in
different solvents. The emission spectra of the DDPC also reveal the intramolecular
charge transfer band character. These findings confirm that there is a significant
electron transfer between the donating moiety and the accepting fragment through the
π conjugated. DDPC dye undergoes solubilization in different micelles and may be
used in the determination of CMC of surfactants (SDS and CTAB). DDPC dye can
also use as probe of the polarity and hydrogen bonding properties of its local
microenvironment. The antibacterial activity of DDPC was examined using
cultures of bacteria and the results showed that DDPC showed better
antibacterial activity for both types of the bacteria (Gram-positive and Gram-
negative) as compared to reference drug tetracycline.
Acknowledgments
The authors are thankful to the Chemistry Department at King Abdulaziz University
for providing research facilities.
16

Page 17 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
References
1. B. K. Srivastava, M. Solanki, B. Mishra, R. Soni, S. Jayadev, D. Valani, M. Jain,
P. R. Patel, Bioorg. Med. Chem. Lett., 2007, 17, 1924.
2. Y. Jiao, B. Xin, Y. Zhang, X. Wu, X. Lu, Y. Zheng, W. Tang, X. Zhou, Eur. J.
Med. Chem., 2015, 90, 170.
3. M. H. Helal, S. A. ElꢀAwdan, M. A. Salem, T. A. Abdꢀelaziz, Y. A. Moahamed,
A. A. ElꢀSherif, G. A. M. Mohamed, Spectrochemica Acta A: 2015, 135, 764.
4. S. Musiu, G. Purstinger, S. Stallinger, R. Vrancken, A. Haegeman, F. Koenen, P.
Leyssen, M. Froeyen, J. Neyts, J. Paeshuyse, Antiviral Res., 2014, 106, 71.
5. R. M. Mohareb, M. Y. Zaki, N. S. Abbas, Steroids, 2015, 98, 80.
6. G. Jose, T. H. S. Kumara, G. Nagendrappa, H. B. V. Sowmya, J. P. Jasinski, S.
P. Millikan, S. S. More, B. Janardhan, B. G. Harish, N. Chandrika, J. Mol.
Struct. 2015, 1081, 85.
7. P. P. Dixit, T. P. A. Devasagayam, S. Ghaskadbi, Eur. J. Pharmacol., 2008, 581,
216.
8. M. Abdallah, I. Zaafarany, S. O. AlꢀKaranee, A. A. ElꢀFattah, Arabian J. Chem.,
2012, 5, 225.
9. P. Dixit, M. P. Khan, G. Swarnkar, N. Chattopadhyay, R. Maurya, Bioorg. Med.
Chem. Lett., 2011, 21, 4617.
10. C. B. Mishra, S. Kumari, M. Tiwari, Eur. J. Med. Chem., 2015, 92, 1
11. D. Zhu, X. Chen, R. Huang, S. Yan, J. Lin, Tetrahedran, 2015, 71, 2363.
12. V. Duros, H. Sartzi, S. J. Teat, Y. Sanakis, O. Roubeau, S. P. Perlepes, Inorg.
Chem. Commun., 2014, 50, 117.
13. A. P. Menezes, A. Jayarama, S. W. Ng, J. Mol. Struct., 2015, 1088, 85.
17

RSC Advances
Page 18 of 28
View Article Online
DOI: 10.1039/C6RA02814D
14. C. S. Stanm, C. Peptu, N. Marcotte, P. Horlescu, D. Sutiman, Inorg. Chim. Acta.,
2015, 429, 160
15. S. Guang, S. Yin, H. Xu, W. Zhu, Y. Gao, Y. Song, Dyes & Pigm., 2007, 73,
285.
16. D. Liaw, K. Wang, S. P. Pujari, Y. Huang, B. Tao, M. Chen, K. Lee, J. Lai,
Dyes & Pigm. 2009, 82, 109.
17. Y. Z. Wang, D. D. Gebler, D. K. Fu, T. M. Swager, A. G. MacDiarmid, A. J.
Epstein, Synth. Met. 1997, 85, 1179.
18. M. Suzuki, Thin Solid Films, 1989, 180, 253.
19. W. Ying, X. Zhang, X. Li, W. Wu, F. Guo, J. Li, H. Agren, J. Hua, Terahedron,
2014, 70, 3901.
20. H. M. Marwani, A. M. Asiri, S. A. Khan, Spectral, stoichiometric ratio,
physicochemical, polarity and photostability studies of newly synthesized
chalcone dye in organized media, J Lumin., 2013, 136, 296.
21. S. K. Lanke, N. Sekar, J Photochem. Photobio A: 2016, 321, 63ꢀ71.
22. K. Ablajan, W. Kamil, A. Tuoheti, S. WanꢀFu, Molecules, 2012, 17, 1860ꢀ
1869.
23. S. A. ElꢀDaly, A. M. Asiri, A. Y. Obeid, S. A. Khan, K. A. Alamry, M. A.
Hussien, G. A. AlꢀSehemi, Opt Laser Technol., 2013, 45, 605.
24. S. A. ElꢀDaly, A. M. Asiri, S. A. Khan, K. A. Alamry, J. Lumin., 2013, 134,
819.
25. R. Pandian, E. Naushad, V. Vijayakumar, G. H. Peters, P. M.
Nanjappagounder, Chem Cent J 2014, 8, 1ꢀ7
26. R. Frolicker, J. Phys. Chem. A: 2002, 106, 1708.
27. S. Suppan, J. Photochem. Photobiol. A:, 1990, 50, 293.
18

Page 19 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
28. N. Mataga, T. Kubota, Molecular interaction and electronic spectra, New York,
Marcel Dekker, 1970, 371.
29. M. Gaber, S. A. ElꢀDaly, T. A. Fayed, Y. S. ElꢀSayed, Opt. Laser Technol.,
2008, 40, 528.
30. C. Reichardt, Solvents and Solvent Effects in Organic Chemistry, 2nd ed., VCH,
Weiheim, 1988: 359.
31. C. Reichardt, C.: Solvents and solvent effects in organic chemistry, 2 nd ed.
Weiheim: VCH, 1988 p.35
.
32. N. J. Turro, Molecular photochemistry (frontiers in chemestry), 1st ed., W. A.
Benjamin, Inc., Reading, MA, 286 (1965)
33. B. J. Coe, J. A. Harris, I. Asselberghs, K. Clays, G. Olbrechts, A. Persoons, J.
T. Hupp, R. C. Johnson, S. J. Coles, M. B. Hursthouse, K. Nakatani, Adv. Funct.
Mater. 2002, 12, 110.
34. K. Rurack, M. L. Dekhtyar, J. L. Bricks, U. ReschꢀGenger, W. Retting
,
J.
Phys. Chem. A 1999, 103, 9626.
35. R. Gahlaut, N. Tewari, J. P. Bridhkoti, N. K. Joshi, H. C. Joshi, S. Pant, J. Mol.
Liq., 2011, 161, 141.
36. P. Carpena, J. Aguiar, P. BernaolaꢀGalvan, and C. C. Ruiz, Langmuir 2002, 18,
6054ꢀ6058.
37. S. A. ElꢀDaly, A. M. Asiri, K. A. Alamry, A. Y. Obaid, J. Lumin., 2013, 139,
69.
38. K. N. Ganesh, P. Mitra, D. Balasubramanian, J. Phys. Chem. 1982, 86, 4291.
39. K. A. Dill, D. E. Koppel, R. S. Cantor, J. D. Dill, D. Bendouch, S. H. Chen,
Nature, 1984, 309, 42.
40. W. Kirk, W. Wessels, Biophys. Chem., 2007, 125, 32.
19

RSC Advances
Page 20 of 28
View Article Online
DOI: 10.1039/C6RA02814D
41. S. A. Khan, A. M. Asiri, K. Sharma, Med. Chem. Res., 2013, 22, 1998.
20

Page 21 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
Table 1: Spectral data of DDPC in different solvents
ꢀ
ν
ε
ꢁ 12
Debye
ET (30)
(cmꢀ1)
Φf
Solvent
M ꢀ1cmꢀ
f
ꢀf
λab(nm)
λem(nm)
ETN
Kcal
1
molꢀ1
EtOH
0.305
1.28
72.38
395
559
8410
0.24
4.48
7427
0.092
DMSO
MeOH
DMF
CHCl3
CH2Cl2
0.266
0.308
0.263
0.217
0.255
1.25
1.32
1.35
1.35
1.33
1.38
1.41
1.41
1.43
71.47
73.49
74.26
74.65
73.87
75.43
76.65
76.44
77.06
400
389
385
383
387
379
373
374
371
548
561
540
532
530
542
490
497
475
11480
8073
9590
9158
7810
9183
11840
8614
9752
0.31
0.25
0.28
0.26
0.21
0.29
0.30
0.22
0.23
5.12
4.53
4.77
4.59
4.14
4.82
4.86
4.17
4.25
6752
7881
7396
7313
6972
7935
6401
6617
5902
0.29
0.035
0.33
0.44
0.40
0.30
0.43
0.34
0.16
Acetonitrile 0.274
Dioxan
THF
CCl4
0.148
0.263
0.024
Table 2. Antibacterial activity of DDPC positive control: Tetracycline and negative control
(DMSO) measured by the Halo Zone Test (Unit, mm) and MIC.
Corresponding effect on microorganisms
A. hydrophila
Y. enterocolitica
L. monocytogenes P. aeruginosa
DDPC ( Disc)
DDPC (MIC)
11.4 ± 0.3
12.5 ± 0.5
14.3 ± 0.2
15.2 ± 0.2
64
64
32
32
Tetracycline (Disc)
Tetracycline (MIC)
DMSO
13.0 ± 0.5
20.0 ± 0.5
12.0 ± 0.5
14.0 ± 0.5
32
ꢀ
32
ꢀ
32
ꢀ
32
ꢀ
21

RSC Advances
Page 22 of 28
View Article Online
DOI: 10.1039/C6RA02814D
0.12
0.10
0.08
0.06
0.04
0.02
0.00
EtOH
DMSO
MeOH
DMF
CHCl3
CH2Cl2
CCl4
CH3CN
Dioxan
THF
350
400
450
500
550
600
Wavelength (nm)
Fig.1ꢀ Electronic absorption spectra of 1 x 10ꢀ5 mol dmꢀ3 of DDPC in different
solvents.
450
400
350
EtOH
MeOH
DMSO
DMF
300
250
200
150
100
50
CHCl3
CH2Cl2
CCl4
CH3CN
Dioxan
THF
0
400
450
500
550
600
650
Wavelength (nm)
Fig.2ꢀ Emission spectra of 1 x 10ꢀ5 mol dmꢀ3 of DDPC in different solvents (λ ex
=
385 nm)
22

Page 23 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
80
75
70
65
60
55
50
45
40
Ea
Ef
35
40
45
50
55
ET(30)kcal mol-1
Fig.3ꢀ Plot of energy of absorption (Ea) and emission (Ef) versus ET(30) of
different solvents
8000
7500
7000
6500
6000
5500
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
ꢀ
f
Fig.4ꢀ Plot Stokes shift (ꢀυ ) versus polarity (∆f) of solvent for DDPC
23

RSC Advances
Page 24 of 28
View Article Online
DOI: 10.1039/C6RA02814D
0.5
0.4
0.3
0.2
0.1
0.0
30
35
40
45
50
55
ET(30) Kcal mol-1
Fig.5ꢀ Plot of
φf versus ET (30) of different solvents
90
80
70
60
50
40
30
20
7
000
0
-4
cmc = 7.25 x 10
M
600
00
50
000
400
00
30
000
200
00
0.00
00
0.00
04
0.00
0.00
12
0.00
16
0.00
08
20
-3
mol dm )
[CTAB]
(
Fig.6ꢀ Plot of If versus the concentration of CTAB
24

Page 25 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
10
0
600
550
500
450
400
350
300
0
0
90
80
70
60
50
40
30
0
0
0
-3
cmc = 7.45 x 10
M
0
0
25
20
15
00
00
00
0.0
00
0.0
04
0.0
08
0.0
12
0.0
16
-3)
[SDS] (mol dm
Fig.7ꢀ Plot of If versus the concentration of SDS
500
450
400
350
300
250
200
150
100
50
(b)
(a)
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0
1
2
3
4
5
6
7
[MeOH] mol dm-3
0
400 425 450 475 500 525 550 575 600 625 650
Wavelength (nm)
Fig. 8. (a) Fluorescence quenching of 1x10ꢀ5 mol dmꢀ3 DDPC in dioxan (
λ
= 385
ex
nm) by MeOH, the concertration of MeOH at decreasing emission intensity are 0,
0.49, 1.47, 2.46, 3.45, 4.43, 5.43, 5.42, 6.41, 7.31 and 8.38 mol dmꢀ3. (b) Stern–
Volmer plot of fluorescence quenching of 1x10ꢀ5 mol dmꢀ3 of DDPC in dioxan by
MeOH
25

RSC Advances
Page 26 of 28
View Article Online
DOI: 10.1039/C6RA02814D
525
450
375
300
225
150
75
(a)
(b)
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0
1
2
3
4
5
[EtOH] mol dm-3
0
400 425 450 475 500 525 550 575 600 625 650
Wavelength (nm)
Fig. 9. (a) Fluorescence quenching of 1x10ꢀ5 mol dmꢀ3 DDPC in dioxan (
λ
= 385
ex
nm) by EtOH, the concentration of EtOH at decreasing emission intensity are 0, 0.34,
1.02, 1.71, 2.39, 3.07, 3.76 and 4.44 mol dmꢀ3. (b) Stern–Volmer plot of fluorescence
quenching of 1x10ꢀ5 mol dmꢀ3 of DDPC in dioxan by EtOH
26

Page 27 of 28
RSC Advances
View Article Online
DOI: 10.1039/C6RA02814D
500
450
400
350
300
250
200
150
100
50
(a)
(b)
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
[PrOH] mol dm-3
0
400 425 450 475 500 525 550 575 600 625 650
Wavelength (nm)
Fig. 10. Fluorescence quenching of 1x10ꢀ5 mol dmꢀ3 DDPC in dioxan by (
λ
= 385
ex
nm) PrOH, the concentration of PrOH at decreasing emission intensity are 0, 0.23,
0.71, 1.18, 1.66, 2.13, 2.61, 3.08 and 3.56 mol dmꢀ3 . (b) Stern–Volmer plot of
fluorescence quenching of 1x10ꢀ5 mol dmꢀ3 of DDPC in dioxan by PrOH
27

RSC Advances
Page 28 of 28
View Article Online
DOI: 10.1039/C6RA02814D
450
400
350
300
250
200
150
100
50
(b)
(a)
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
[BuOH] mol dm-3
0
400
450
500
550
600
650
Wavelength (nm)
Fig. 11 (a). Fluorescence quenching of 1x10ꢀ5 mol dmꢀ3 DDPC in dioxan (
λ
= 385
ex
nm) by BuOH, the concentration of BuOH at decreasing emission intensity are 0, .21,
0.65, 1.08, 1.52, 1.95, 2.39, 2.82 and 3.25 mol dmꢀ3 (b) Stern–Volmer plot of
.
fluorescence quenching of 1x10ꢀ5 mol dmꢀ3 of DDPC in dioxan by BuOH
28