Synthesis and fluorescence study of 6,7-diaminocoumarin and its imidazolo derivatives

Article abstract of DOI:10.1016/j.dyepig.2012.08.021

Novel 6,7-diamino-4-methylcoumarin and derived imidazolocoumarins were synthesized and their absorption and fluorescence properties were recorded in solvents of varying polarity. 6,7-Diamino-4-methylcoumarin is highly fluorescent, with a quantum yield of

Full text of DOI:10.1016/j.dyepig.2012.08.021

Dyes and Pigments 96 (2013) 525e534
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Dyes and Pigments
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Synthesis and fluorescence study of 6,7-diaminocoumarin and its imidazolo
derivatives
*
T. Sheshashena Reddy, A. Ram Reddy
Department of Chemistry, University College of Science, Osmania University Campus, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 6,7-diamino-4-methylcoumarin and derived imidazolocoumarins were synthesized and their
absorption and fluorescence properties were recorded in solvents of varying polarity. 6,7-Diamino-4-
methylcoumarin is highly fluorescent, with a quantum yield of 0.78 in dioxane. The fluorescence
intensity of 6,7-diamino-4-methylcoumarin, when mixed with different carbonyl group containing
compounds, decreased. Based on the interaction of this diaminocoumarin with the carbonyl compound
a fluorescent sensor with high selectivity towards electron rich benzaldehydes and cinnamaldehyde was
developed.
Received 28 March 2012
Received in revised form
16 August 2012
Accepted 19 August 2012
Available online 27 August 2012
Keywords:
6,7-Diamino-4-methylcoumarin
Fluorescence
Ó 2012 Elsevier Ltd. All rights reserved.
Solvatochromism
Quenching
Quantum yield and Derivatization
1. Introduction
benzaldehydes. Therefore, in this paper we report the synthesis and
photophysical properties of hitherto unreported VI and four of its
Coumarins comprise a group of natural compounds found in
a variety of plant sources. Coumarins are attractive fluorescent
molecules due to their extended spectral range, high emission
quantum yields, photostability and good solubility in common
solvents. As a consequence of these features coumarins are widely
used as laser dyes [1e3]. Typical features of these derivatives can be
readily modified by introduction of substituents in the chromene
ring as well as in the annulated benzene ring, imparting flexibility
for various applications including as laser dyes [4], fluorescence
markers [5], fluoride ion sensor [6], cyanide ion sensor [7,8] and
metal ion sensors [9e12]. 2,3-Diaminonaphthalenes were
employed as nitric oxide sensors [13,14]. There is increasing
evidence that unsaturated aldehydes generated endogenously
during the degradation process of biological molecules, such as
lipid peroxidation, glycation and amino acid oxidation, are involved
in the onset and progression of much pathology such as cardio-
vascular atherosclerosis, long-term complications of diabetes and
neurodegenerative diseases [15,16]. Owing to the immense
importance of coumarin dyes in the sensor field and the endoge-
nous production of aldehydes, we have developed 6,7-diamino-4-
methylcoumarin (VI) as a sensor for the detection of selected
imidazolo derivatives. The multistep linear synthesis is initiated
from 3-aminophenol. The absorption and fluorescence properties
of these compounds were studied in ten solvents of different
polarity and the relative quantum yields were evaluated employing
9,10-diphenylanthracene as the standard. The photophysical
properties of VI are drastically altered in presence of small amounts
of a few select aromatic aldehydes having electron donating
substituents at either the ortho- or para position and the
a,b-
unsaturated aldehyde, cinnamaldehyde.
2. Experimental
2.1. Materials
Spectroscopic grade organic solvents were obtained from Finar
Chemicals. Starting and other chemicals and reagents were
purchased from SigmaeAldrich unless otherwise stated and were
used without further purification. Thin layer chromatography (TLC)
was performed on silica gel 60 F254 aluminum plates (Merck).
2.2. Instrumentation
The melting points reported were uncorrected and determined
in Polmon instrument (model no. MP-96). The IR spectra were
* Corresponding author. Tel.: þ91 9849684195; fax: þ91 4027090020.
E-mail address: a_ramreddy@yahoo.com (A. Ram Reddy).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2012.08.021

526
T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
recorded on Bruker Infrared model Tensor-27. 1H NMR and 13C NMR
were recorded on a Bruker 300 MHz Ultrashield spectrometer. The
EI mass spectra were recorded on a VG micro mass 7070-H. The
LCMS spectra were recorded on LCMS 2010, Shimadzu, Japan. LC
conditions were used C18 column, mobile phase methanol: water
(90:10 v/v) and at UVevis wavelength 254 nm. In the mass
spectrum ESI technique is used. UVevis spectra were recorded on
Elico SL 159 UVevis spectrophotometer. Steady state fluorescence
1H NMR (300 MHz, DMSO-d6):
exchanged), 8.31 (s, 1H, aromatic), 6.80 (s, 1H, aromatic), 6.15 (s,
1H), 2.36 (s, 3H, eCH3). IR (KBr): nmax/cmꢁ1 3350 (
NH), 1726 ( CO).
Mass spectral data: m/z ¼ 221(M þ 1). Elemental analysis (found C
54.38, H 3.37, N 12.61% C10H8N2O4 required C 54.55, H 3.66, N
12.72%).
d
¼ 7.74 (s, 2H, NH2, D2O
n
n
2.3.5. Synthesis of 6,7-diamino-4-methyl-2H-chromen-2-one (VI)
6-Nitro-7-amino-4-methyl-coumarin (1 g, 4.54 m mol) was
heated under reflux for 4 h with (0.89 g, 6.81 m mol) of tin in conc.
hydrochloric acid (15 mL). After cooling, the mixture was poured in
to water (50 mL) and let stand overnight. The resulting suspension
was made slightly basic with 50% NaOH with cooling by addition of
ice chips and extracted with ethyl acetate (3 ꢂ 100 mL). All the
organic layers were combined and dried over sodium sulphate.
Ethyl acetate was removed under vacuum to get yellow solid. Yield
(0.2 g, 23.5%), m.p. 237e241 ꢀC. Yellow color, 1H NMR (300 MHz,
was
investigated
on
Shimadzu
RF-5301PC
spectro-
fluorophotometer with 5 nm excitation and emission slit widths at
25 ꢀC employing 1 cm path length quartz cell. Elemental compo-
sition was determined by elemental analyzer, Elementar, Vario EL
model. The pH measurements were carried out on a Global digital
pH meter.
2.3. Synthesis of imidazolocoumarins
2.3.1. Synthesis of 3-carbethoxyaminophenol (II)
DMSO-d6):
aromatic), 6.48 (s, 1H, aromatic), 5.80 (s, 1H), 4.88(brs, 2H, NH2, D2O
exchanged), 2.23 (s, 3H, eCH3). 13C (75 MHz, DMSO-d6):
¼ 161.9,
154.1, 148.5, 141.7, 132.1, 109.7, 107.9, 99.5, 18.52. IR (KBr): nmax/cmꢁ1
3390 ( NH), 1677 (
d
¼ 7.72 (s, 2H, NH2, D2O exchanged), 6.76 (s, 1H,
Ethylchloroformate (0.9 mL, 9.17 mmol) was added in one
portion to a stirred suspension of m-aminophenol (1 g, 9.17 mmol)
in ethyl acetate (15 mL). A white precipitate formed immediately.
The reaction mixture was stirred for 2 h at room temperature. The
amine hydrochloride precipitate was removed by filtration. Ethyl
acetate was removed under reduced pressure to obtain colorless
crystals of 3-carbethoxyaminophenol (1.4 g, 84.6%). m.p. 94e96 ꢀC
[17].
d
n
n
CO). Mass spectral data: m/z ¼ 191(M þ 1).
Elemental analysis found (C 62.94, H 5.22, N 14.45% C6H6 N2O2
required C 63.15, H 5.30, N 14.73%).
2.3.6. Synthesis of 8-methylchromeno[7,6-d]imidazol-6(3H)-one
(VIIa)
2.3.2. Synthesis of 7-carbethoxyamino-4-methylcoumarin (III)
3-Carbethoxyaminophenol (2 g, 11.04 mmol) and ethyl-
acetoacetate (1.70 mL, 13.25 mmol) were suspended in 70% sulfuric
acid (30 mL) and were stirred at room temperature for 4 h. The
solution was poured in to an iceewater mixture (100 mL) resulting
in the formation of a precipitate which was collected and crystal-
lized from ethanol to give 7-carbethoxyamino-4-methylcoumarin
6,7-Diamino-4-methyl-coumarin (0.1 g, 0.526 mmol) was
heated under reflux for 4 h in formic acid (10 mL). After cooling, the
mixture was poured in to water (30 mL) and the resulting
suspension was made slightly basic with the addition of 5% NaHCO3
with cooling by addition of ice chips. The TLC pure brown precip-
itate was filtered and washed with ice water. Yield (76 mg, 73%),
m.p. >300 ꢀC. Yellow color, 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.19
(2.26 g, 83%). 1H NMR (300 MHz, CDCl3):
d
¼ 8.78 (s, 1H, eNH),
(s, 1H), 7.99 (s, br, 1H, eNH), 7.65 (s, 1H, aromatic), 7.48 (s, 1H,
6.79 (d, 1H, Jmeta ¼ 7.4 Hz), 6.66 (d,, 1H, Jmeta ¼ 2.2 Hz), 6.54 (dd,, 1H,
Jortho ¼ 7.4 Hz, Jmeta ¼ 2.2 Hz), 5.58 (s, 1H), 3.86 (q, 2H, eOCH2), 2.33
(s, 3H, eCH3), 1.40 (t, 3H, eCH3). mp 185e187 ꢀC [17].
aromatic), 6.18 (s, 1H), 2.56 (s, 3H, eCH3). 13C (75 MHz, DMSO-d6):
d
¼ 160.2, 155.0, 153.4, 149.2, 140.9, 136.5, 114.4, 111.6, 110, 100.8,
18.7. IR (KBr): nmax/cmꢁ1 3367 (
n
NH), 1720 ( CO). Mass spectral
n
data: m/z ¼ 201(M þ 1). Elemental analysis (found C 65.82, H 4.10,
2.3.3. Synthesis of 6-nitro-7-carbethoxyamino-4-methylcoumarin
(IV)
N 13.73% C11H8 N2O2 required C 66.00, H 4.03, N 13.99%).
Aluminum nitrate nonahydrate (1.29 g, 1.5 mmol) was added
portionwise to 7-carbethoxyamino-4-methylcoumarin (1 g,
4.04 mmol) in acetic anhydride (25 mL) over a 10 min period and
stirred at room temperature for overnight. After 12 h the reaction
mixture was poured in to ice cold water (100 mL). The resulting
yellow precipitate was filtered and washed with water. The solid
was further purified by column chromatography employing hex-
ane:ethyl acetate (9:1 v/v). Yield (0.54 g, 45.7%), m.p. 162e164 ꢀC.
2.3.7. Synthesis of 2,8-dimethylchromeno[7,6-d]imidazol-6(3H)-
one (VIIb)
Following the above method, compound VIIb was prepared by
heating under reflux in acetic acid in place of formic acid. Yield
(77 mg, 69%), m.p.>300 ꢀC. Yellow color, 1H NMR (300 MHz, DMSO-
d6):
aromatic), 6.13 (s, 1H), 2.52 (s, 3H, eCH3), 2.44 (s, 3H, eCH3). 13C
(75 MHz, DMSO-d6):
¼ 160.5, 155.4, 153.6, 149, 140.8, 136.2, 114.6,
111.9, 110, 100.5, 18.7, 14.7. IR (KBr): nmax/cmꢁ1 3362 (
NH), 1726
d
¼ 7.92 (s, br, 1H, eNH), 7.71 (s, 1H, aromatic), 7.32 (s, 1H,
d
Yellow color, 1H NMR (300 MHz, CDCl3):
d
¼ 10.08 (s, 1H, eNH),
n
8.59 (s, 1H, aromatic), 8.48 (s, 1H, aromatic), 6.28 (s, 1H), 4.31 (q, 2H,
(
n
CO). Mass spectral data: m/z ¼ (M þ 1) 215. Elemental analysis
eOCH2), 2.47 (s, 3H, eCH3), 1.38 (t, 3H, eCH3). IR (KBr): nmax/cmꢁ1
(found C 67.13, H 4.47, N 12.92% C12H10 N2O2 required C 67.28, H
4.71, N 13.08%).
3318 (nNH) 1729 (nCO), 1716 (nCO). Mass spectral data: m/
z ¼ (M þ 1) 293.
2.3.8. Synthesis of 8-methyl-2-phenylchromeno[7,6-d]imidazol-
6(3H)-one (VIIc)
2.3.4. Synthesis of 6-nitro-7-amino-4-methyl-2H-chromen-2-one
(V)
6,7-Diamino-4-methyl-coumarin (0.02 g, 0.105 m mol) was
taken in DMF (5 mL) solvent, to this benzaldehyde (0.012 g,
0.105 m mol) was added and was heated under reflux for 4 h. After
cooling, the DMF was removed under reduced pressure. The
resulting residue was washed with hexane (20 mL) to obtain yellow
colored product. Yield (18 mg, 65%), m.p. >300 ꢀC. 1H NMR
Deprotection of IV was carried out by heating 6-nitro-7-
carbethoxyamino-4-methylcoumarin (1 g, 3.42 mmol) under
reflux for 4 h in a mixture of ca. H2SO4 (3.05 g) and acetic acid
(2.87 g). After cooling, the mixture was poured in to water (50 mL)
and let stand overnight. The resulting suspension was made slightly
basic with 50% NaOH with cooling by addition of ice chips. The
yellow precipitate was filtered and washed with ice water
(2 ꢂ 30 mL). Yield (0.65 g, 86.5%), m.p. >300 ꢀC. Dark yellow color,
(300 MHz, DMSO-d6):
6.29 (s, 1H), 2.54 (s, 3H, -CH3). IR (KBr): nmax/cmꢁ1 3360 (
CO). Mass spectral data: m/z ¼ 277.28 (M þ 1). Elemental analysis
d
¼ 8.14e8.15 (m, 2H), 7.53e7.60 (m, 5H),
n
NH), 1726
(n

T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
527
(found C 73.64, H 4.17, N 10.03% C17H12 N2O2 required C 73.90, H
4.38, N 10.14%).
the presence of acetic anhydride to give 6-nitro-7-aminoprotected-
4-methylcoumarin, IV. Its structure was confirmed by melting
point, proton NMR and mass spectra. In the proton NMR spectrum
disappearance of C-6 proton and appearance of singlet signals at
8.60 and 8.49 ppm confirms formation nitro derivative. The signal
at 8.60 ppm was assigned to the proton ortho to the nitro group.
6-Nitro-7-protectedaimno-4-methylcoumarin, IV was heated
under reflux in acetic acid and conc. sulfuric acid mixture to
deprotect the amino group. The deprotection was confirmed by its
proton NMR spectrum. In the proton NMR spectrum, the amine
protons were observed at 7.80 ppm, C-5 and C-8 protons gave
singlet signals at 8.37 and 6.85 ppm, respectively. Disappearance of
the ethyl group proton signals in the aliphatic region indicated the
formation of 6-nitro-7-amino-4-methyl-2H-chromen-2-one, V.
6-Nitro-7-amino-4-methylcoumarin, V was treated with Sn, HCl
at 100 ꢀC to give 6,7-diamino-4-methylcoumarin, VI. The structure
of 6,7-diamino-4-methylcoumarin was confirmed by NMR and IR
spectroscopy and mass spectrometry and microanalysis. In the
proton NMR spectrum broad singlet signals at 7.81 ppm and
4.93 ppm are due to two amino group protons attached to C6 and
C7. The C-8 and C-5 carbon attached protons gave singlet signals at
6.57 ppm and 6.84 ppm, respectively. The C-3 attached proton gave
a singlet signal at 5.80 ppm and C-4 carbon attached methyl
protons gave a singlet signal at 2.23 ppm. In the 13C NMR spectrum,
the lactone carbonyl carbon gave a signal at 161.9 ppm, C-3 carbon
at 154.1, the coumarin oxygen adjacent carbon gave a signal at
148.5. The amino group ipso carbon atoms gave signals at 141.7 and
132.1 ppm. In the mass spectrum, the molecular ion peak observed
2.3.9. Synthesis of 8-methyl-2-styrylchromeno[7,6-d]imidazol-
6(3H)-one (VIId)
6, 7-Diamino-4-methyl-coumarin (0.02 g, 0.105 m mol) was
taken in DMF (5 mL) solvent, to this cinnamaldehyde (0.013 g,
0.105 m mol) was added and was heated under reflux for 4 h. After
cooling, the DMF was removed under reduced pressure. The
resulting residue was washed with hexane (20 mL) to obtain dark
yellow colored product. Yield (21 mg, 68%), m.p. >300 ꢀC. 1H NMR
(300 MHz, DMSO-d6):
7.46e7.24 (m, 7H), 6.29 (s, 1H), 2.52 (s, 3H, eCH3). IR (KBr): nmax
cmꢁ1 3358 (
NH), 1724 (
d
¼ 12.94 (s, 1H, NH), 7.75e7.68 (m, 2H),
/
n
nCO). Mass spectral data: m/z ¼ 303.28
(M þ 1). Elemental analysis (found C 75.19, H 4.42, N 9.05% C19H14
N2O2 required C 75.48, H 4.67, N 9.27%).
3. Results and discussion
7-Aminocoumarin is a bright fluorescent molecule. However on
annulation with a pyrrole ring at 7,8-positions its fluorescence was
enhanced significantly [18]. As the VI is a diaminocoumarin, it is
expected to be fluorescent and a potential reagent for the deter-
mination of carbonylic compounds [19].
3.1. Synthesis of VI and imidazolocoumarin
A variety of synthetic methodologies are known for the
synthesis of benzimidazoles, e.g. the condensation of ortho phe-
nylenediamine (OPDA) with either benzaldehydes or benzoic acids
[20,21]. In the present study VI is considered as an analogue of
OPDA and its synthesis was carried out following Scheme 1.
Subsequently, the VI was condensed with acetic acid, formic acid,
benzaldehyde and cinnamaldehyde to yield the respective imida-
zolocoumarins in good yields.
3-Aminophenol was treated with ethylchloroformate to give the
protected aminophenol, ethyl-3-hydroxyphenylcarbamate, II. Its
melting point of 94e96 ꢀC coincided with the literature value [17].
Compound II was further treated with ethylacetoacetate to give the
protected amino coumarin III. Compound III was characterized by
melting point, proton NMR and mass spectra. In the 1H NMR
spectrum of III, the coumarin characteristic C3-H proton was
observed as a singlet signal at 5.58 ppm. The singlet signal at
2.33 ppm was assigned to the methyl protons attached to C-4. The
coumarin III was nitrated with aluminum nitrate nonahydrate in
at 191 [M
methylcoumarin.
þ
1] indicates the formation of 6,7-diamino-4-
6,7-Diamino-4-methylcoumarin, VI was treated with formic
acid, acetic acid, benzaldehyde and cinnamaldehyde separately to
yield respective imidazole derivatives, VII(aed). In the 1H NMR
spectrum of VIIa, the imidazole ring carbon attached proton was
observed as a singlet signal at 8.19 ppm, imidazole ring nitrogen
attached proton was observed at 7.99 as a broad singlet. The C-9, C-
4 and C-7 carbon attached protons appeared as singlets at 7.65, 7.48
and 6.18 ppm, respectively. The methyl protons attached to
coumarin gave a singlet at 2.56 ppm. In the 13C NMR spectrum of
VIIa, the lactone carbonyl carbon gave a signal at 160.2 ppm, C-2
carbon at 155.0, C-8 carbon at 153.4 and coumarin oxygen adjacent
carbon gave a signal at 149.2. The amino attached ipso carbons gave
signals at 140.9, 136.5 ppm. In the mass spectrum of VIIa,
a molecular ion peak observed at 201 [M þ 1] and indicates the
formation of imidazolocoumarin VIIa.
Scheme 1. Synthesis of 6,7-diamino-4-methylcoumarin and its imidazole derivatives.

528
T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
In the 1H NMR spectrum of VIIb, the imidazole ring methyl
protons were observed as a singlet at 2.52 ppm and in its mass
spectrum, a molecular ion peak at m/z 215 [M þ 1] indicates the
formation of imidazolocoumarin, VIIb.
wavelength absorption maxima of VI, VIIa, VIIb, VIIc and VIId are
varying in the range of 326e389 nm, 307e329 nm, 321e327 nm,
340e346 nm and 360e374 nm, respectively. It can be noticed
that the electronic absorption maxima of imidazolocoumarins, VII,
depend on the substituents present in the imidazole part of the
molecule. This may be due to the change in the basic absorbing
chromophore unit. The conversion of VI to imidazolocoumarin
brings a change in the electronic transition from coumarin chro-
mophore to benzimidazole chromophore. Compound VIId has
absorption maxima in between 360 nm and 374 nm in various
solvents. The absorption maximum at longer wavelength in VIId
may be due to the extended styryl chromophore moiety while there
is no such extended chromophore is present in VIIa and its
absorption maximum observed was between 307 nm and 329 nm
in different solvents. Compound V is an immediate precursor of VI
and has a nitro group in a complimentary position which resulted
in the absorption maximum shifting to a longer wavelength than
the latter. The solvent polarity and the hydrogen bond between the
solvent and coumarin make the absorption wavelength occurring
at longer wavelength while the hydrogen bond between the
solvent and the amino nitrogen atom of imidazole ring has an
opposite effect on the position of the long wavelength absorption
maximum.
In the 1H NMR spectrum of VIIc, the imidazole substituted
phenyl protons were observed as a mutiplet at 7.53e7.60 ppm and
in its mass spectrum, a molecular ion peak at m/z 277 [M þ 1]
indicates the formation of imidazolocoumarin, VIIc. Similarly when
everything being equal in the 1H NMR spectrum of VIId, the imid-
azole substituted phenyl protons resonated as a multiplet at 7.46e
7.24 ppm and the two ethylinic protons signal was merged in the
aromatic multiplet signal at 7.46e7.24 ppm. Further the structure of
VIId was established by its molecular ion peak at m/z 303 [M þ 1].
The mass spectra of VII clearly indicates that the final product is
imidazolocoumarin and not the dihydroimidazolocoumarin or
imine. A possible mechanism for the formation of imidazolocou-
marin is given in Scheme 2. Hidenori et al. reported that when
OPDA was reacted with benzaldehyde the intermediate dihy-
droimidazole that was obtained unstable and it was readily con-
verted to benzimidazole by aerial oxidation [22e24]. It is further
confirmed by the LC-MS studies that when the LC-MS of the
mixture of VI and benzaldehyde and VI and cinnamaldehyde was
taken the former gave a peak at m/z 277 [M þ 1] and the later gave
a peak at m/z 303 [M þ 1], respectively indicating that the corre-
sponding dihydroimidazole was not present.
3.3. Fluorescence study
3.2. Electronic absorption spectral study
7-Aminocoumarins are the most important subset of coumarins
with wide spread applications as fluorescent probes [25,26].
Coumarin fluorescent probes exhibit significant solvatochromism
and has been an area of intense investigation for almost 40 years.
The main aim of the present study is to investigate the photo-
physical and photochemical properties of VI, VI and subsequently
its utility in the detection of carbonyl containing compounds. VI is
a structural analogue of ortho phenylenediamine (OPDA) and can
In Fig. 1 the electronic absorption spectra of VI in six different
solvents are shown. From Fig. 1 it can be noticed that the longer
wavelength absorption maximum of VI in chloroform solvent is at
326 nm and in dimethylsulfoxide it is red shifted to 389 nm
exhibiting solvatochromism. The spectral data obtained in the
electronic absorption studies are given in Table 1.The longer
Scheme 2. Possible mechanism for the formation of benzimidazole ring.

T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
529
point oxidant/solvent) [32], PhI(OAc)2 [33], 2,3-dichloro-5,6-
dicyanobenzoquinone (DDQ) [34], NaHSO3 [35], H2O2/HCl [36],
and Na2S2O5 [37], air [24], sulfamic acid [38], FeCl3$6H2O [39],
Sc(OTf)3 [40], KHSO4 [41], TsOH/graphite and N,N-dimethylaniline/
graphite [42].
The effect of solvent polarity on the photophysical properties of
VI and VII was investigated in different organic solvents of varying
polarity. Table 1 (supplementary information tables, Tables S1, S2)
summarizes the spectral characteristics, i.e., absorption maxima
1. Chloroform
2. Dichloromethane
3. Acetonitrile
4. Methanol
5. Isopropanol
0.30
0.25
0.20
0.15
0.10
0.05
0.00
4
2
3
1
6. Dimethylsulphoxide
6
(
lA), excitation maxima (lex.max), fluorescence maxima (lflu.max),
Stokes shift (Dn (cmꢁ1)) and fluorescence quantum yield (V) of VI,
VIIa, VIIb, VIIc and VIId in various solvents.
5
The relative fluorescence quantum yield,
F
is evaluated by
employing Eq. (1) [43]:
300
350
400
450
500
ꢀ
ꢁꢀ
ꢁꢀ
ꢁ
2
Wavelength (nm)
Iunk
Istd
Aunk
Astd
hunk
hstd
Funk
¼
Fstd
(1)
Fig. 1. The UVevisible absorption spectra of VI [2.75 ꢂ 10ꢁ5 M] in different solvents at
25 ꢀC.
where
F, Fstd, Iunk, Istd, Astd, Aunk, hunk and hstd are the fluorescence
quantum yields, the integral of the emission intensities, the
absorbance at the excitation wavelength and the refractive indexes
of the corresponding solvents of the unknown samples and the
standard, respectively. 9,10-Diphenylanthracene was used as stan-
readily yield benzimidazole system on treatment with a carboxyl
compound. The synthesis of 2-substituted benzimidazoles involves
the treatment of 1,2-phenylenediamines either with carboxylic
acids [27] or their derivatives (nitriles, imidates, or orthoesters)
[28], under acidic conditions and sometimes combined with very
high temperatures (i.e., polyphosphoric acid, 180 ꢀC) or the use of
microwave irradiation [29]. The benzimidazoles were also often
generated from the condensation of phenylenediamines with
aldehydes [30] under oxidative conditions [31] using various
oxidative and catalytic reagents, such as nitrobenzene (high-boiling
dard (F
¼ 0.9) [43].
Compound VI is highly fluorescent while its precursors are less
fluorescent. The fluorescence spectra of VI in different solvents are
shown in Fig. 2. From Fig. 2, it can be observed that the VI exhibited
a single emission maximum in all solvents except in dichloro-
methane. The single emission maximum in chloroform is at 413 nm
and occurs at shorter wavelength when compared to the remaining
solvents. In dichloromethane, two emission maxima were ob-
tained. Among these two emission maxima, one is characteristic to
that obtained in chloroform at 413 nm and the other is character-
istic to that obtained in the remaining solvents at longer wave-
length, at about 500 nm. Similar to the 7-aminocoumarin, VI with
two amino groups experienced a strong red-shift in polar solvents
and correlate well with the hydrogen bonding. The H-bond
acceptor nucleophilic solvents also caused a strong red-shift in the
emission maximum of VI. This can be attributed to the preferential
solvation in the excited state [44]. From Table 1 it can be observed
that the Stokes shift vary in between 5285 cmꢁ1 in chloroform and
7800 cmꢁ1 in water (pH 7.4). In polar protic solvents, the Stokes
shift was maximum. This is largely due to the occurrence of fluo-
rescence emission maxima at longer wavelengths. In polar aprotic
solvents like DMSO, acetone and acetonitrile the emission maxima
Table 1
Spectral properties of VI and imidazolocoumarins.
Compound Solvent
number
labs (nm) lex(nm) lem (nm) Stokes shift Ff
Dn (cmꢁ1
)
VI
1,4-Dioxane
Chloroform
Ethyl acetate
Dichloromethane 345
366
326
366
370
339
369
340
370
370
378
378
379
387
368
468
413
474
419
483
485
502
498
497
498
516
5660
5285
6003
5545
6324
6409
6675
6375
6265
5759
7800
0.783
0.526
0.550
0.697
0.535
0.463
0.265
0.383
0.338
0.488
0.140
Acetone
373
367
375
378
378
389
368
Acetonitrile
Methanol
Ethanol
Isopropanol
DMSO
Water (pH7.4)
VIIc
1,4-Dioxane
Chloroform
Ethyl acetate
Dichloromethane 346
Acetone
Acetonitrile
Methanol
Ethanol
Isopropanol
DMSO
344
345
343
348
345
349
346
349
349
348
348
346
353
345
404
388
417
389
417
417
421
421
419
421
424
3984
3213
4672
3195
4672
4672
4900
4900
5036
4576
5401
0.097
0.260
0.090
0.193
0.101
0.104
0.261
0.283
0.276
0.165
0.182
340
341
343
343
342
346
345
Water (pH7.4)
VIId
1,4-Dioxane
Chloroform
Ethyl acetate
Dichloromethane 367
Acetone
Acetonitrile
Methanol
Ethanol
Isopropanol
DMSO
360
369
365
363
360
367
370
368
369
366
365
364
373
364
418
429
423
431
422
423
422
422
420
430
430
3625
4460
3607
3825
3477
3607
3425
3700
3663
3554
4429
0.156
0.015
0.262
0.018
0.284
0.244
0.225
0.247
0.231
0.492
0.205
368
367
366
366
364
374
364
Water (pH7.4)
Fig. 2. The fluorescence spectra of VI [2.75 ꢂ 10ꢁ5 M] in different solvents at 25 ꢀC.

530
T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
also occurred at longer wavelength. But in the case of chlorinated
solvents, chloroform and dichloromethane, the emission maxima
were blue shifted. The fluorescence quantum yield of VI varied
between 0.14 in water (pH 7.4) and 0.78 in dioxane solvent. The
fluorescence quantum yield of VI in polar protic solvents like water
(pH 7.4), methanol, ethanol and isopropanol is diminished and
appears at 0.14, 0.265, 0.383 and 0.338, respectively. This can be
attributed to an increased nonradiative decay rate in protic solvents
[45]. The order of fluorescence quantum yield in the solvents
investigated is 1,4-dioxane > DCM
protic solvents, there is an increased reorientation of solvent
molecules around the dye through hydrogen bonding. On the other
hand, in proton accepting polar aprotic solvents, VI in its exited and
ground state is stabilized by hydrogen bonding interactions via its
amino groups. The emission spectral shifts of VI are large when
compared to its absorption spectral shifts with change in polarity of
the solvent. The smaller shift in the absorption spectra and the
larger shifts in the emission clearly indicate that the dipole moment
of the exited state is higher compared to that of ground state
leading to the preferential solvation of exited state over the ground
state [44].
> acetone > ACOEt > CHCl3>DMSO > AN > EtOH > iso-
propanol > methanol > water (pH 7.4). A similar observation was
made in the case of 7-aminocoumarin.
3.4. Detection of aldehydes
The interactions between the solvent and solute affect the
energy difference between the ground and excited states. Lipperte
Mataga equation provides the relationship between the spectral
shift and the solvent polarity [46] [Eq. (2)].
The effect of different aldehydes, ketones, esters and acids on
the fluorescence of VI was studied in methanol at 25 ꢀC without
addition of catalyst and heating. The aldehydes used were benz-
aldehyde, benzaldehydes with electron donating and electron
withdrawing groups at ortho and para positions, cinnamaldehyde
and retinal. The ketones used were acetophenone and acetone.
The ester employed was ethyl acetate and the acids used were
acetic acid and formic acid. Based on the spectral results, the
carbonyl containing compounds that interacted with VI were
categorized into three types. The first categories of carbonyl con-
taining compounds are those that can shift the emission
maximum of VI to longer wavelength and quench its fluorescence.
These include all the aliphatic aldehydes, acetophenone and
retinal. They quench the fluorescence between 1.06 and 1.99 times
as shown in Table 2. The quenching ratio was obtained by dividing
the fluorescence quantum yield of VI with the fluorescence
quantum yield of VI in presence of respective carbonyl containing
compounds. The second category of carbonyl containing
compounds are substituted benzaldehydes with an electron
withdrawing group at either the ortho or para or ortho and para
positions. These shifted the fluorescence emission maxima of VI
from 502 nm to lower wavelength but not below 480 nm. From
ꢀ
ꢁ
2
hc
3
ꢁ 1
h2 ꢁ 1
ð
mE
ꢁ
mGÞ2
nA
ꢁ
nF
¼
ꢁ
þ Const
(2)
h2 þ 1
a3
2
3
ꢁ 1
2
where h is Planck’s constant, C is the speed of light, and a is the
radius of the cavity in which the fluorophore resides. nA and nF are
the absorption and emission maxima in cmꢁ1
,
h
is the refractive
index and
3
is the dielectric constant, respectively. The first
parentheses in Eq. (2), is difference of two terms ((
3
ꢁ 1)/(2 þ 1)
3
and (h2 ꢁ 1)/(2h2 þ 1)), the difference of these terms accounts for
the spectral shifts due to reorientation of the solvent molecules and
hence called the orientation polarizability (Df) Eq. (3).
ꢀ
ꢁ
3
ꢁ 1
h2 ꢁ 1
D
f ¼
ꢁ
(3)
h2 þ 1
2
3
ꢁ 1
2
In order to determine the solvatochromic behavior of VI, a plot
was drawn between Dn nA nF) and polarity parameter f. The
(
ꢁ
D
LipperteMataga plot is given in Fig. 3. From Fig. 3 it can be observed
that the relationship between the Stokes shift and the polarity
parameter is non-linear. The non-linear relationship between the
Stokes shift and the polarity implies that the spectral shifts are
dependent on specific solvent solute interactions like H-bonding.
VI has functional groups which can act as H-acceptor and H-
donor. Therefore, the LipperteMataga correlation followed two
distinctive lines, one for protic and one for non-protic solvents. In
Table
2 it can be observed that these carbonyl containing
compounds quenched the fluorescence quantum yield of VI by
1.90e7.80 times. The third categories of carbonyl containing
compounds are benzaldehyde, benzaldehyde derivatives with
electron donating substituents and cinnamaldehyde. These
quenched the fluorescence quantum yield of VI by 3.27e24.09
times. Examination of Table 2 reveals that benzaldehyde and
cinnamaldehyde have a remarkable effect on the fluorescence of
VI and had prompted for further investigation.
From Fig. 4A it can be observed that addition of benzaldehyde
(3.28 ꢂ 10ꢁ3 M) to the methanolic solution of VI (2.75 ꢂ 10ꢁ5 M),
the emission and excitation wavelengths of the latter had shifted
from 502 nm and 377 nm to 421 nm and 347 nm, respectively. The
possible reason for the substantial quenching of fluorescence and
blue shift of excitation and emission wavelengths of VI in the
1.1,4-dioxane
2.Chloroform
3.Ethyl acetate
4.Dichloromethane
5.DMSO
6800
6700
6600
6500
6400
6300
6200
6100
6000
5900
5800
5700
5600
5500
5400
5300
5200
y = 8099.x + 4050
R² = 0.996
10
7
6
9
6.Isopropanol
7.Ethanol
8
presence of
a few selected benzaldehydes required detailed
8.Acetone
9.Acetonitrile
10.Methanol
examination. In an ongoing effort to understand the mechanism of
the profound blue shift and quenching of the fluorescence of VI,
synthesis of imidazolocoumarins was undertaken as depicted in
Scheme 1. Compounds phenylimidazolocoumarin, VIIc and styr-
ylimidazolocoumarin, VIId are the imidazolocoumarins derived
from VI on reaction with benzaldehyde and cinnamaldehyde. From
Fig. 4A it is evident that, the wavelength of fluorescence emission
maxima of VIIc and VI in presence of benzaldehyde [1.64 ꢂ 10ꢁ3 M]
are coincident (Tables 1 and 2). Therefore it can be considered that
VI, when mixed with benzaldehyde, readily upon irradiation at its
absorption maximum is converted to an imidazolocoumarin which
is less fluorescent than VI. The formation of phenyl-
imidazolocoumarin, VIIc in the spectroscopic solution comprising
y = 15879x + 1579
R² = 0.997
4
1
5
3
2
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Orientation polarizability
Fig. 3. The LipperteMataga plot for VI.

T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
531
Table 2
Spectral property of VI on addition of aldehydes in methanol.
Entry
Compound VI þ aldehyde
Solvent
lex (nm)
lem (nm)
Stokes shift
V
Vf of diamine/Vf diamine þ aldehydes
1
2
3
4
5
6
7
8
Nil
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
378
354
365
367
360
374
368
386
378
408
406
400
374
409
409
376
413
412
412
412
414
413
412
416
380
378
378
378
502
423
434
423
419
438
421
424
497
485
485
502
483
487
484
502
502
504
502
502
504
507
505
506
501
502
502
502
6675
4900
4390
3600
3910
3906
3421
3425
6334
3890
4012
5079
6034
3915
3787
6674
4292
4430
4350
4350
4313
4489
4350
4275
6355
6675
6675
6675
0.265
0.011
0.081
0.072
0.064
0.018
0.071
0.013
0.04
0.053
0.139
0.124
0.034
0.041
0.046
0.224
0.218
0.187
0.203
0.226
0.219
0.186
0.147
0.133
0.057
0.240
0.248
0.248
e
Benzaldehyde
24.09
3.27
3.68
4.14
14.72
3.73
20.38
6.62
5.0
1.90
2.13
7.79
6.46
5.76
1.18
1.21
1.41
1.30
1.17
1.21
1.42
1.80
1.99
4.64
1.10
1.06
1.06
2-Metyl Benzaldehyde
3-Metyl Benzaldehyde
4-Metyl Benzaldehyde
4-Methoxy Benzaldehyde
4-isopropyl benzaldehyde
Cinnmaldehyde
4-Choloro Benzaldehyde
2-Choloro Benzaldehyde
2,4-dichlorobenzaldehyde
4-Nitrobenzaldehyde
4-fluorobenzaldehyde
2-fluorobenzaldehyde
2-bromobenzaldehyde
Acetaldehyde
Propanal
Butanal
Pentanal
Hexanal
Heptanal
Octanal
Isoverlaldehyde
Cyclohexyl-1-al
Furfuraldehyde
Retinal
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Acetophenone
Acetic Acid
VI and benzaldehyde was further supported by the LC-MS data. The
LC-MS of the spectroscopic solution of VI and benzaldehyde is
shown in Fig. 5. From Fig. 5 it can be noticed that the retention time
4.13 min (methanol: water (80:20 v/v)) correspond to m/z/277
(M þ 1) in the mass spectrum which indicates the formation of VIIc
in the spectroscopic solution. The LC-MS of the spectroscopic
solution of VI and benzaldehyde and VIIc in other mobile phase
(methanol:water (90:10 v/v)) the retention time was 33 s while
VIIc has exhibited a retention time of 33.5 s which correspond to
m/z/277 (M þ 1) in the mass spectrum. Similar phenomena were
observed with the other substituted benzaldehydes possessing
electron donating substituents.
Fig. 4. A. Fluorescence spectra of VI-benzaldehyde system in methanol at 25 ꢀC a). VI [2.75 ꢂ 10ꢁ5 M] lex 378 nm b). VI [2.75 ꢂ 10ꢁ5 M] þ 3.28 ꢂ 10ꢁ3 M benzaldehyde lex 347 nm
c). VIIc [1.35
ꢂ
10ꢁ5 M] lex 347 nm. B: Fluorescence spectra of VI-cinnamaldehyde system in methanol at 25 ꢀC. a). VI [2.75
ꢂ
10ꢁ5 M] lex 378 nm b). VI
[2.75 ꢂ 10ꢁ5 M] þ cinnamaldehyde 3.20 ꢂ 10ꢁ3
M
lex 366 nm c). VIId [3.20 ꢂ 10ꢁ5 M] lex 366 nm.

532
T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
Fig. 5. LC-MS of binary mixture of VI and benzaldehyde in methanol solution.
In continuation and to confirm the chemical derivatization, the
excitation and emission wavelengths. Spectrum e of Fig. 6 shows
the quenched and altered fluorescence spectrum of the benzal-
dehyde [1.64 ꢂ 10ꢁ3 M] e VI [2.20 ꢂ 10ꢁ5 M] physical mixture. At
this concentration of benzaldehyde [1.64 ꢂ 10ꢁ3 M], the fluores-
cence emission maximum of VI occurred was at 424 nm and with
further increase in benzaldehyde concentration the fluorescence
intensity increased experiencing a little blue shift. However, at
[3.28 ꢂ 10ꢁ2 M] of benzaldehyde, the new emission maximum
attained maximum intensity and the additional amount of benz-
aldehyde led to the quenching of perspective imidazolocoumarin,
respectively. The isoemissive point observed at 454 nm and the
emission curves from a to f in Fig. 6 reveals that VI is gradually
effect of cinnamaldehyde on the fluorescence of VI was investigated.
In Fig. 4B, the effect of cinnamaldehyde on the fluorescence spectra
of VI is shown. From Fig. 4B it can be observed that with addition of
cinnamaldehyde, the fluorescence of VI was quenched significantly
with profound blue shift in its emission maximum. The fluorescence
quenching of VI and gradual appearance of a new band at lower
wavelength with the disappearance of emission maximum at
502 nm with increased cinnamaldehyde is similar to that of the
effect of benzaldehyde. Spectrum c in Fig. 4A and B is the emission
spectrum
of
the
phenylimidazolocoumarin
and
styr-
ylimidazolocoumarin. The emission maxima and the excitation
maxima of these two imidazolocoumarins are coinciding with the
emission and excitation maxima of VI in presence of benzaldehyde
[3.28 ꢂ 10ꢁ3 M] and cinnamaldehyde [3.20 ꢂ 10ꢁ3 M] in solution
(Fig. 4). It is interesting to note that when acetaldehyde was added to
VI and the emission and excitation spectra of the physical mixture
were taken, there was no similarity between the emission spectra of
latter with that of methylimidazolocoumarin. It is interesting to note
that the emission and excitation spectra of the physical mixture of
acetaldehyde and VI have no similarity with the emission and
excitation spectra of methylimidazolocoumarin (VIIb). On the other
hand the fluorescence emission maximum obtained for VI-acetal-
dehyde binary mixture was at 502 nm, no change in the emission
wavelength. The quenching of fluorescence of VI in the presence of
benzaldehyde and cinnamaldehyde is due to the formation of imi-
dazolocoumarins while there is no such covalent derivatization
occurred with the aliphatic aldehydes and benzaldehydes possess-
ing electron withdrawing substituents. Ester and carboxylic acids
have no effect on the fluorescence spectrum of VI.
In Fig. 6 the effect of concentration of benzaldehyde on the
fluorescence of VI is given. From Fig.
6 it is clear that,
Fig. 6. Fluorescence response of VI inꢁm5 ethanol to benzaldehyde at 25 ꢀC a. VI
3.28 ꢂ 10ꢁ3 M, 1.64 ꢂ 10ꢁ3 M, 6.56 ꢂ 10ꢁ2 M and 9.84 ꢂ 10ꢁ2 M benzaldehyde.
1.64 ꢂ 10ꢁ3 M concentration of benzaldehyde is the minimum
[2.20
ꢂ
10ꢁ5 M], beg. VI [2.20
ꢂ
10
M]
þ
3.28
ꢂ
10ꢁ2 M, 1.64 ꢂ 10ꢁ2 M,
concentration at which the phenomenon of shifting in the

T. Sheshashena Reddy, A. Ram Reddy / Dyes and Pigments 96 (2013) 525e534
533
converted to phenylimidazolocoumarin with addition of benzal-
750000
700000
650000
600000
550000
500000
450000
400000
350000
300000
dehyde and these two have identical fluorescence intensity at
450 nm. But at greater than [3.28 ꢂ 10ꢁ2 M] of benzaldehyde, the
VI has been completely and irreversibly converted to phenyl-
imidazolocoumarin. The Rf value of the compound formed in the
solution is found to be identical with the Rf value of VIIC (on Merck
silica gel 60 F254 aluminum plates using chloroform:methanol
(9.5:0.5 v/v) as eluent).
y=1826.13x+355320
R2=0.997
The mechanism of fluorescence quenching of VI with the third
category of carbonyls is due to the formation of imidazolocou-
marins. It was further noticed that when the electron donating
groups are substituted at ortho position and or if the electron
donating group is a poor electron donor like methyl group, the
rate of formation of imidazolocoumarin is slow. In Fig. 7, the time
course for the rate of quenching of fluorescence of VI in presence
of benzaldehyde and 4-methyl benzaldehyde is provided. From
Fig. 7 it can be observed that when the rate of diminishing of
fluorescence of VI was monitored at a 5 s time interval, the rate of
quenching of fluorescence of VI in presence of benzaldehyde is
faster than that in presence of para tolualdehyde. A similar trend
was observed with other less effective electron donating groups
like isopropyl, meta tolualdehyde and ortho tolualdehyde. This was
further confirmed by the second order rate constants, k2. Under
the condition [VI] ¼ [aldehyde], plots of [1/A] vs. time were linear
with a positive slope and an intercept on the ordinate indicating
that the reaction follows overall second-order kinetics. In Fig. 8,
a typical plot between 1/[A] vs t (s) is shown. [A] is the concen-
tration of VI and was derived from the fluorescence emission
intensity in presence of respective aldehyde. The k2 values ob-
0
50
100
150
200
Time (sec)
Fig. 8. Second order kinetics plot for the fluorescence quenching of VI in presence of
benzaldehyde.
4. Conclusion
Akin to the 7-aminocoumarin, the new 6,7-diamino-4-
methylcoumarin, VI is highly fluorescent. It exhibits remarkable
solvatochromism and its fluorescence is sensitive to a set of alde-
hydes. In the presence of benzaldehyde, benzaldehydes with elec-
tron donating groups and cinnamaldehyde, VI developed to
imidazolocomarins which are less fluorescent. The decreased
fluorescence by the formation of derivatives leads to their selective
detection by fluorometry.
tained were 1826.13 Mꢁ1Sꢁ1, 457 Mꢁ1Sꢁ1, 399 Mꢁ1Sꢁ1, 85 Mꢁ1Sꢁ1
,
43.66 Mꢁ1Sꢁ1, 36.50 Mꢁ1Sꢁ1 and 29.36 Mꢁ1Sꢁ1 benzaldehyde,
cinnamaldehyde, 4-methoxy benzaldehyde, 4-methyl benzalde-
hyde, 4-isopropyl benzaldehyde, 3-methyl benzaldehyde and 2-
methyl benzaldehyde respectively. The rate of fluorescence
quenching with the unsubstituted benzaldehyde was faster than
all the aldehydes investigated. The next efficient quenching
occurred with cinnamaldehyde. Everything being equal, the
benzaldehydes having electron donating group at para position
was faster than the electron donating group at ortho position.
Greater is the electron donating ability faster is the quenching. The
reason for the faster formation of imidazolocoumarins, especially
with benzaldehyde and cinnamaldehyde over the other ring
activating groups is not immediately known. Therefore, VI is
a selective fluorophore that can yield a covalent derivative with
a few selected benzaldehydes at different rates of time intervals
leading to its fluorescence quenching.
Acknowledgements
We wish to gratefully acknowledge the financial support from
Department of Biotechnology, Ministry of Science and Technology,
Government of India. One of the authors T. Sheshashena Reddy
thanks CSIR, New Delhi for the award of senior research fellowship
and Dr. K. Varaprasad Rao for providing the starting material for the
synthesis of diaminocoumarin.
Appendix A. Supplementary data
Supplementary data related to this article can be found in the
online version at http://dx.doi.org/10.1016/j.dyepig.2012.08.021.
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