Synthesis and Photophysical Studies on Naphthalimide Derived Fluorophores as Markers in Drug Delivery

Article abstract of DOI:10.1007/s10895-016-1835-y

Derivatives of 4-amino-1,8-naphthalimide containing a free alkyl chain bearing carboxyl group as linker and different substituents at 4-amino function have been synthesized, characterized and studied for their photophysical properties. Steady state fluorescence studies showed quantum yield varied from 0.45 to 0.65 with Stokes shift in the range of 5824–8558?cm^?1. Spectroscopic and physicochemical parameters, like electronic absorption, emission, and extinction coefficient were investigated in order to explore the analytical potential of compounds. Solvatochromic studies demonstrated that all compounds were sensitive towards the polarity of different solvents showing the highest degree of fluorescence in acetonitrile. In addition, the compounds in the presence of ions, viz. Na⁺, K⁺ and Mg²⁺ at concentration of 0.1–2 equivalents, showed a decreasing trend in fluorescence with increasing ionic concentration. TCSPC set – up was used to measure the fluorescence lifetime of compounds, which was found to be bi-exponential with longer and shorter component at their respective amplitudes. The average lifetime of compounds was observed to be 5.76–9.96?ns indicating the possibility of their greater utilization in research and diagnosis.

Full text of DOI:10.1007/s10895-016-1835-y

J Fluoresc
DOI 10.1007/s10895-016-1835-y
ORIGINAL ARTICLE
Synthesis and Photophysical Studies on Naphthalimide Derived
Fluorophores as Markers in Drug Delivery
Nidhi Singh1 & Ritika Srivastava1 & Anuradha Singh1 & Ramendra K. Singh1
Received: 9 March 2016 /Accepted: 9 May 2016
#
Springer Science+Business Media New York 2016
Abstract Derivatives of 4-amino-1,8-naphthalimide contain-
ing a free alkyl chain bearing carboxyl group as linker and
different substituents at 4-amino function have been synthe-
sized, characterized and studied for their photophysical proper-
ties. Steady state fluorescence studies showed quantum yield
varied from 0.45 to 0.65 with Stokes shift in the range of 5824–
8558 cm−1. Spectroscopic and physicochemical parameters,
like electronic absorption, emission, and extinction coefficient
were investigated in order to explore the analytical potential of
compounds. Solvatochromic studies demonstrated that all com-
pounds were sensitive towards the polarity of different solvents
showing the highest degree of fluorescence in acetonitrile. In
addition, the compounds in the presence of ions, viz. Na+, K+
and Mg2+ at concentration of 0.1–2 equivalents, showed a de-
creasing trend in fluorescence with increasing ionic concentra-
tion. TCSPC set – up was used to measure the fluorescence
lifetime of compounds, which was found to be bi-exponential
with longer and shorter component at their respective ampli-
tudes. The average lifetime of compounds was observed to be
5.76–9.96 ns indicating the possibility of their greater utiliza-
tion in research and diagnosis.
Introduction
The use of fluorescent molecules as reporter groups for label-
ing of biomolecules, like nucleic acids, proteins, etc. is a very
vital and dynamic research field [1]. The synthesis of
fluorophores possessing selectivity, sensitivity and desirable
properties is always a great challenge. 1,8-Naphthalimide unit
is a special class of widely accepted fluorescent probe [2, 3].
Owing to good properties, like strong fluorescence, good
photostability and electroactivity, these compounds have
found major applications in different areas, such as antitumor
and anticancer research [4], fluorescent markers in biochem-
istry [5], high performance intramolecular charge transfer
based electroluminescent materials [6, 7], light emitting di-
odes [8], analgesics in medicine [6], optical brighteners in
detergent and textiles [9], fluorescence switchers [10], liquid
crystal additives [11], photo induced electron transfer based
sensors [12], laser dyes [13],etc. The 1, 8- naphthalimide
shows a major variation in wavelength at emission when a
polar group (electron donating or withdrawing) is present at
C-4 position because these groups affect the delocalization of
electron density inside the rings [14, 15]. 1, 8-Naphthalimide
possessing yellow fluorescence has been reported as a fluo-
rescent probe in hypoxic cells [16, 17]. Thus, design and syn-
thesis of new compounds based on 1, 8-naphthalimide unit
have gained importance in modern day researches in science
and technology.
.
Keywords Amino-naphthalimide derivative
.
.
Solvatochromism Quantum yield Steady state
.
fluorescence Average lifetime
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-016-1835-y) contains supplementary material,
which is available to authorized users.
Here, we report some newly designed 4-amino-1,8-
naphthalimide derivatives, 3–6, having different substituents
at C-4 amino function and an alkyl chain bearing carboxylic
group for attachment with any suitable molecule. The fluores-
cence of these molecules has been compared against our ear-
lier reported molecule 2 used for oligonucleotide labelling
[18–20]. Effect of different solvents and ions on fluorescence
properties of these molecules along with their average
* Ramendra K. Singh
rksinghsrk@gmail.com
1
Bioorganic Research Laboratory, Department of Chemistry,
University of Allahabad, Allahabad 211002, India

J Fluoresc
fluorescence lifetime has been reported. Some other fluores-
cent molecules based on 4-amino-1,8-naphthalimide have
been used previously in our laboratory for labelling and study-
ing their effect on hybridization of oligodeoxyribonucleotides
involving Tm measurement and gel shift assay [21–23].
solutions of compounds were prepared in 10 mL of volumetric
flask using acetonitrile/water (2:8, v/v). The 20 μL of prepared
stock solution was further diluted to 2 mL by addition of the
same solvent mixture. The prepared solutions of metal ions
were 0.1 equivalent of the compound concentration.
All calculations, like the area under curve for calculation of
quantum yield, calculation of molar absorptivity, smoothening
and processing of graph at observed value of absorption and
emission were performed using Origin Pro 8.1. IBH DAS
(Version 6) decay analysis software was used to calculate
fluorescence lifetime with pre-exponential factor throughout
the present study.
Experimental
Chemicals and Reagents
Acenaphthene, aminocaproic acid, acetyl chloride, p-
toluenesulphonyl chloride, di-tert-butyl-dicarbonate, ethyl
chloroformate, methane sulphonyl chloride were purchased
from Sigma Aldrich Chemicals Pvt. Ltd., New Delhi. Other
reagents and solvents (AR grade) were used without further
purification.
Synthesis and Characterization
6-(6-Amino-1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)
-hexanoic acid (1)
Apparatus
Acenaphthene (64.9 mmol, 10 g) was dissolved in hot glacial
acetic acid (80 mL) and cooled gradually upto 10 °C to form a
crystalline magma. Nitric acid (6 mL) was added drop wise to
this magma for 20–25 min. While maintaining the temperature
at 10–15 °C. Now the temperature was allowed to rise to 30–
35 °C and stirred the reaction mixture for another 30 min. The
mixture was poured into crushed ice with constant stirring to
make uniform mixture, filtered out the residue obtained and 5-
nitroacenaphthene was purified through column chromatogra-
phy using hexane and ethyl acetate as eluent. Yield 64.2 %
(8.3 g); M.p.: 102 °C; Rf 0.7 (EtOAc:Hexane; 2:8); UV
(MeOH): 370 nm.
To a stirred solution of sodium dichromate (100 mmol,
6.2 g) and acetic acid (20 mL), 5-nitroacenaphthene (40 mmol,
8.0 g) was added gradually. The reaction mixture was then
refluxed on oil-bath for 5 h. The reaction mixture was cooled
and transferred to cold water followed by filtration to give the
orange precipitate. The precipitate was purified by column
chromatography to obtain 4-nitro-1,8-naphthalic anhydride
using hexane and ethyl acetate as eluent. Yield 63 % (6.2 g);
M.p.: 228 °C; Rf 0.5(EtOAc:Hexane; 4:6); UV (MeOH):
340 nm; 1H NMR (CDCl3): δ 8.93–8.95 (m, 1H), 8.73–8.79
(m, 2H), 8.45–8.46 (m, 1H), 8.05–8.09 (m, 1H).
To a stirred solution of 4-nitro-1,8-napthalic anhydride
(16.4 mmol, 4.0 g) in ethanol (30 mL) was added the solution
of SnCl2.H2O (49.2 mmol, 10 g) in concentrated HCl (10 mL)
at room temperature. The reaction mixture was refluxed for
12 h. After ensuring complete reduction, the reaction mixture
was cooled down to room temperature and aqueous solution
of Na2CO3 (10 %) was added to quench the reaction. The
precipitate was collected by filtration, washed with water
and dried in vacuo to afford the reddish orange crude product
4-amino-1,8-naphthalic anhydride, which was directly used
All reactions were monitored by TLC using Merck 60 F254
precoated silica gel plates (0.25 mm thickness) and the prod-
ucts were visualized by UV detection. Flash chromatography
was carried out with silica gel (200–300 mesh). FT-IR spectra
were recorded on a Bruker Tensor-27 spectrometer. 1H NMR
spectra were recorded on a Bruker Avance (III) 400 MHz
spectrometer. Data for 1H NMR are reported as chemical shift
(δ ppm), multiplicity (s = singlet, d = doublet, q = quartet,
m = multiplet), coupling constant J (Hz), integration and as-
signment. Melting points were recorded on a Thomas Hoover
capillary melting apparatus without correction. The solvents
used were spectroscopic grade. The absorption spectra were
taken on Varian UV-Vis spectrophotometer (model: Cary
100). Emission Spectra were taken on fluoromax-4p fluorim-
eter from Horiba Yovin (Model: FM-100). The fluorescence
spectra were corrected for spectral sensitivity of the instru-
ment. The excitation and emission slits were 2/2 nm for the
emission measurements. All measurements were done at
25 °C. For the time resolved studies, a picosecond time cor-
related single photon counting (TCSPC) system from Horiba
Yovin (Model: Fluorocube-01-NL) was used. The samples
were excited at 375 nm using a picosecond diode laser (model:
Pico Brite-375 L). The repletion rate was 5 MHz. The signals
were collected at magic angle (54.70) polarization using a
photomultiplier tube (TBX-07C) as the detector, which has a
dark count of 20 cps. The instrument response function of the
setup was ~140 ps. The data analysis was done using IBH
DAS (version 6) decay analysis software.
The effect of ions on intensity of absorption and emission
spectra were recorded in aqueous media. Since the com-
pounds were insoluble in 100 % aqueous media, stock

J Fluoresc
Scheme 1 General method for
synthesis of compound 1
for further synthesis. Yield 50 % (2 g), M.p.: 358 °C; Rf
0.6(DCM:MeOH; 9.5:0.5); UV (MeOH): 430 nm.
chromatography using DCM and MeOH as eluent. Yield
66 % (2 g); M.p.: 320 °C; Rf 0.7(DCM:MeOH; 2:8); UV
(MeOH): 430 nm, 260 nm.
To a stirred solution of 6-aminocaproic acid (13.1 mmol,
1.75 g, 1.4 eq) in pyridine (6 mL) was added 4-amino-1,8-
naphthalic anhydride (9.4 mmol, 12 g). Reaction mixture was
then stirred at 80–85 °C for 3–4 h and monitored by TLC. After
completion of reaction, cooling the reaction mixture at room
temperature, followed by evaporation of organic solvent, left
the crude product, which was purified by column
6-(6-Acetylamino-1,3-dioxo-1H,3H-benzo[de]
isoquinolin-2-yl)-hexanoic acid (2)
To a stirred solution of compound 1 (0.61 mmol, 200 mg) in
small amount of pyridine/acetonitrile (1:1, v/v) acetyl chloride
Scheme 2 Synthetic route for
compounds 2–6

J Fluoresc
MeOH
Table 1 Absorption and
emission maxima of compounds
2–6 in different solvents
Compound
Absorbance λabs (nm)
Emission λem (nm)
CHCl3
CH3CN
EtOH
MeOH
CHCl3
CH3CN
EtOH
2
3
4
5
6
345
339
338
402
406
347
405
335
410
413
346
428
339
422
432
346
424
340
425
433
436
511
429
516
499
454
530
430
533
514
461
548
470
551
531
465
565
440
555
532
(70 μL, 1.5 eq) was added drop wise. The reaction mixture
was stirred for 6 h. After completion of the reaction
(monitored by TLC), solvents were removed in vacuo
and the crude product was purified by column chroma-
tography using DCM and MeOH as eluent to give
125 mg of pure compound 2. Yield 55 % (125 mg);
M.p.: 163 °C; Rf 0.7 (DCM:MeOH; 9.5:0.5); UV
(MeOH): 345 nm; IR (KBr): ν 3077, 3033, 2942,
2864, 1856, 1703, 1660, 1624, 1583, 1520, 1496,
(MeOH): 422 nm; IR (KBr): ν 3431, 3342, 3238, 2923, 2853,
1732, 1695, 1620, 1579, 1524, 1478, 1386, 1360,
1
1320 cm−1; H NMR (DMSO-d6 + Methanol-d4): δ 8.49–
8.57 (m, 3H), 8.23–8.35 (m, 1H), 7.63–7.83 (m, 1H), 4.30
(q, J = 7.24 Hz, 2H), 4.11–4.17 (m, 2H), 1.66–1.75 (m, 4H),
1.45–1.48 (m, 2H), 1.38 (t, J = 7.2 Hz, 3H). MS (ESI): m/z
(M+) 398.15.
6-(6-Tert-butoxycarbonylamino-1,3-dioxo-1H,3H-benzo[de]
isoquinolin-2-yl)-hexanoic acid (4)
1 4 6 4 , 1 4 3 7 , 1 4 0 9 , 1 3 8 6 , 1 3 4 0 , 1 2 6 1 c m − 1
;
1HNMR(CDCl3): δ 11.14 (brs, 1H), 7.88–8.34 (m, 4H), 7.57
(s, 1H), 3.69 (s, 2H), 2.12–2.94 (m, 8H), 1.83 (s, 3H). MS
(ESI): m/z (M+) 368.14.
To an ice-cooled stirred solution of compound 1 (0.61 mmol,
200 mg) in small amount of DCM (3 mL) containing a trace of
pyridine, added di-tert-butyl dicarbonate (210 μL, 1.5 eq)
dropwise and the reaction mixture was allowed to stir for
2 h. After completion (monitored by TLC), evaporation of
the organic solvent left the crude product, which was purified
by column chromatography using DCM and MeOH as eluent.
Yield 52 % (135 mg), M.p.: 178 °C; Rf 0.6 (DCM:MeOH;
9.5:0.5), UV(MeOH): 338 nm; IR (KBr): ν 3393, 2926, 2855,
1777, 1741, 1703, 1662, 1588, 1535, 1510, 1458, 1391,
6-(6-Ethoxycarbonylamino-1,3-dioxo-1H,3H-benzo[de]
isoquinolin-2-yl)-hexanoicacid (3)
To an ice-cooled stirred solution of compound 1 (0.61 mmol,
200 mg) in small amount of DCM (3 mL) containing a trace of
pyridine, added ethyl chloroformate (86 μL, 1.5 eq) drop wise
and the reaction mixture was allowed to stir for 2 h. After
completion (monitored by TLC), evaporation of the organic
solvent left the crude product, which was purified by column
chromatography using DCM and MeOH as eluent. Yield 57 %
(140 mg), M.p.: 185 °C; Rf 0.64 (DCM:MeOH; 9.5:0.5), UV
1
1367 cm−1; H NMR (DMSO-d6): δ 8.53–8.79 (m, 2H),
8.16–8.37 (m, 1H), 7.78–7.94 (m, 2H), 4.01 (brs, 1H), 3.49–
3.56 (m, 2H), 2.96–3.03 (m, 2H), 1.54–1.61 (m, 6H), 1.23 (s,
9H). MS (ESI): m/z (M+) 426.18.
Fig. 1 Solvatochromism of compounds 2–6 in CH3CN

J Fluoresc
Table 2 Photophysical properties of compounds 2–6 in CH3CN
Compound
λabs (nm)
λem (nm)
ε (M−1 cm−1
)
ϕ
Stokes Shift (cm−1
)
Fluorescence Lifetimes Ti [ns]
Average Lifetime [ns]
2
3
4
5
6
347
405
335
410
413
454
530
430
533
514
1.1 × 104
1.0 × 104
1.0 × 104
0.8 × 104
1.2 × 104
0.015
0.51
0.62
0.45
0.65
6792
5824
6595
5629
4758
1.46 (0.23), 9.11 (0.77)
3.96 (0.14), 9.29 (0.86)
1.28 (0.37), 8.40 (0.63)
4.10 (.06), 9.06 (0.94)
1.39 (0.08), 10.7 (0.92)
7.32
8.54
5.76
8.74
9.96
* Recorded at concentration of 10−4 mol/L in CH3CN
6-(6-Methanesulphonylamino-1,3-dioxo-1H,3H-benzo[de]
isoquinolin-2-yl)-hexanoic acid (5)
180 mg, 1.5 eq.) and the reaction mixture was allowed to stir
for 5 h. After completion (monitored by TLC), the crude prod-
uct was extracted with DCM, washed with brine and dried
over anhydrous Na2SO4. The evaporation of the organic sol-
vent left the crude product, which was purified by column
chromatography over silica gel using DCM and MeOH as
eluent. Yield 52 %(162 mg ), M.p.: 180 °C; Rf 0.53
(DCM:MeOH; 9.5:0.5); UV (MeOH): 433 nm; IR (KBr): ν
3433, 3346, 3239, 2924, 2853, 1731, 1695, 1651, 1621, 1580,
1524, 1505, 1479, 1405, 1385, 1360 cm−1; 1H NMR(DMSO-
d6-Methanol-d4): δ 8.45–8.80 (m, 3H), 7.82–7.91 (m, 2H),
7.10–7.40 (m, 3H), 6.83–6.85 (m, 1H), 6.01 (brs, 1H), 3.29–
3.38 (m, 2H), 2.79 (s, 3H), 2.61–2.63 (m, 2H), 2.22–2.24 (m,
3H), 1.89–1.95 (m, 3H). MS (ESI): m/z (M+) 480.14.
To an ice-cooled stirred solution of Compound 1 (0.61 mmol,
200 mg) in small amount of DCM (3 mL) containing a trace of
pyridine, added methanesulfonyl chloride (75 μL, 1.5 eq)
drop wise and the reaction mixture was allowed to stir for
2 h. After completion (monitored by TLC), evaporation of
the organic solvent left the crude product, which was purified
by column chromatography using DCM and MeOH as eluent.
Yield 52 % (130 mg), M.p.: 168 °C; Rf 0.58 (DCM:MeOH;
95:05), UV (MeOH): 422 nm; IR (KBr): ν 3431, 3345, 3239,
2923, 2853, 1733, 1699, 1655, 1620, 1580, 1525, 1479, 1405,
1
1386, 1360, 1310, 1261 cm−1; H NMR( Methanol-d4):δ
8.61–8.71 (m, 2H), 7.86–8.41 (m, 2H), 6.72–7.32 (m, 1H),
4.22 (brs, 1H), 3.70–3.73 (m, 2H), 3.41 (s, 3H), 2.54–2.56 (m,
4H), 2.35–2.43 (m,2H), 1.87–1.88 (m,2H). MS (ESI): m/z
(M+) 404.10.
Results and Discussion
Synthesis
6-[1,3-Dioxo-6-(toluene-4-sulfonylamino)-1H,3H-benzo[de]
isoquinolin-2-yl]-hexanoic acid (6)
The base molecule 1 was prepared in several steps starting
from acenaphthene (Scheme 1) and compounds 2–6 were pre-
pared by substitution reaction at precursor 1 having free amine
group. The compound 2 has already been synthesized and
studied extensively in our laboratotry. In order to improve
To an ice-cooled stirred solution of Compound 1 (0.61 mmol,
200 mg) in small amount of DCM (3 mL) containing a trace of
pyridine, added p-toluene sulfonyl chloride (0.94 mmol,
Fig. 2 Effect of monovalent ionic concentration on fluorescence of compounds 2–6

J Fluoresc
Fig. 3 Effect of bivalent ionic concentration on fluorescence of
compounds 2–6
Fig. 4 Fluorescence decay and lifetime of compounds 2–6 in CH3CN
(ICT) state, resulting in large dipole moment and better stabi-
lization through polarity of solvents in the excited state. The
substituted compounds at position 4 show a clear electronic
donor character for the carboxyimide group and favor the
radiative deactivation of the excited state of the molecules.
Any steric interaction destabilizes the excited state, and thus
causes emission at lower wavelength with decreased radiative
rate constant [24–27]. However, substituent groups at the ami-
no function alter the fluorescence property of this molecule.
and elaborate the fluorescence properties of compound 2, we
synthesized the compounds 3, 4, 5, and 6 by replacing acetyl
group at 4-amino by ethoxycarbonyl, tertiarybutoxycarbonyl,
methane sulphonyl and p-toluene sulphonyl groups (Scheme
2). Compounds 2–6 were purified by silica gel column chro-
matography and characterized by 1H NMR and FT-IR.
Photophysical Properties
In 1,8-naphthalimide having the naphthalene ring and a
dicarboxamide (−CO-N-CO-) group in a six-membered ring,
the excitation involves charge shift away from the electron
donating nitrogen moiety at the 4-position to electron
accepting carbonyl moiety and shows internal charge transfer
Absorption and Fluorescence Spectra
The UV–Vis absorption and emission spectra of compounds
were studied in solvents, like CHCl3, CH3CN, EtOH, MeOH
of different polarity [28, 29]. The absorption and emission
Table 3 Average fluorescence
compound
Solvent
Lifetime (ns)
Relative amplitude
Average Lifetime [ns]
lifetime and relative amplitude
of compounds 2–6 in different
solvents, viz. CHCl3, CH3CN
and MeOH
T1
T2
A1
A2
2
3
4
5
6
CHCl3
CH3CN
MeOH
CHCl3
CH3CN
MeOH
CHCl3
CH3CN
MeOH
CHCl3
CH3CN
MeOH
CHCl3
CH3CN
MeOH
1.39
1.46
1.27
4.70
3.96
1.67
0.80
1.28
0.97
4.70
4.10
2.05
0.82
1.39
0.94
8.84
9.11
0.51
0.23
0.45
0.11
0.14
0.85
0.96
0.37
0.73
0.11
0.06
0.62
0.79
0.08
0.44
0.49
0.77
0.55
0.89
0.86
0.15
.034
0.63
0.27
0.89
0.94
0.38
0.21
0.92
0.56
5.03
7.32
4.75
9.46
8.54
2.47
0.98
5.76
2.53
9.82
8.74
3.53
2.82
9.96
4.64
7.60
10.05
9.30
7.10
5.98
8.40
6.75
10.05
10.00
5.96
10.41
10.71
7.56

J Fluoresc
studies on each compound in different solvents, like CHCl3,
CH3CN, EtOH, MeOH have been shown in Table 1 (graphical
representation can be found as supplementary material).
Further, the absorption and emission spectra of these com-
pounds in CH3CN have been shown in Fig. 1.
marginal. These observations reveal that molecules retained
their fluorescence even in inorganic media and hence could be
an excellent choice as fluorophores to be used for studying
biological system.
All compounds exhibited an intensive electronic absorp-
tion band in the region 345–435 nm. Compounds showed
solvatochromism, meaning thereby a red shift was observed
in the absorption and fluorescence maxima with increasing
dielectric constant of solvents. This indicated that the mole-
cules were better stabilized in their ground and excited states
in more polar solvent, like EtOH, MeOH. Usually polar mol-
ecules have п-п* electronic transitions and higher dipole mo-
ments in the Frank Condon excited state S1 than ground state
S0. The emission spectra of each compound also revealed that
emission occurred at higher wavelength. This change in be-
haviour from non-polar to polar solvents was due to stabiliza-
tion of excited state through polarity of solvent and solvent
effect as all molecules had a capability to form hydrogen bond
(through N and O atoms). The increasing order of wavelength
in emission spectra of these compounds was 4 < 2 < 6 < 3 ≈ 5,
which indicated that excited states of compounds 3 and 5 were
more stabilized and hence, showed red shift. The electron
donating nature of –NH group to naphthalimide ring depends
on the nature of substituents attached and this is expressed in
the degree of fluorescence shown by these compounds Thus
the order of fluorescence of these compounds is
6 > 4 > 3 ≈ 5 > 2. The quantum yield of compounds 2–6
was measured using rhodamine 6G in ethanol as a reference
and showed that electron releasing function at 4-amino group
enhanced the fluorescence signal. Important characterstics of
fluorescent compounds, viz. stokes shift ( A- f), quantum
yield, (Q), absorption (λmax), emission (λmax), fluorescence
lifetime Ti [ns], average lifetime [ns] have been studied for
compounds 2–6 and summarized in Table 2.
Measurement of Fluorescence Lifetime
The time correlated single photon counting (TCSPC) tech-
nique was used to analyse the nature of compounds 2–6 in
the excited state and measure the fluorescence lifetime.
Analyses were performed in solvents of different polarity, like
CHCl3, CH3CN and MeOH where each compound showed
bi-exponential behaviour of lifetime in different solvents at
relative amplitude in excited state (Table 2). Fluorescence de-
cay was measured at the respective emission wavelength of
the molecules 2–6 in CH3CN. The larger and shorter compo-
nents of fluorescence decay along with the respective ampli-
tude and average lifetime of these molecules have been shown
in Table 3 (the fluorescence life time spectra of compounds in
three different solvents can be found as supplementary mate-
rial). The observation revealed that decrease in the value of
lifetime is facilitated by increase in polarity of the solvent. In
general, the solvent polarity affected the molecules in the ex-
cited state resulting in different structural and conformational
changes in the molecule and thus generating other state for
further deactivation [30–32]. Increase in polarity of the medi-
um leads to a shortening of the lifetime because of increase in
non-radiative decay as the energy gap between ground and
relaxed excited state is reduced or sometimes hydrogen bond-
ing becomes a dominating factor in excited state between
molecules and polar solvent. Compound 2 showed longer
component of lifetime 9.11 ns at highest pre-exponential fac-
tor 0.77, compound 4 showed longer component of lifetime
8.40 at pre-exponential factor 0.63, compound 5 showed lon-
ger component of lifetime 10.00 at highest pre-exponential
factor 0.94, compound 6 showed longer component of life-
time 10.71 at highest pre-exponential factor 0.92 in CH3CN
and compound 3 showed longer component of lifetime
10.05 ns at the highest pre-exponential factor 0.89 in chloro-
form. The average lifetime decay of compounds 2–6 has been
shown in Fig. 4.
Fluorescence in Inorganic Media
In order to study the effect of ions on intensity of absorption
and emission spectra, the titration experiments were per-
formed at a definite concentration of prepared stock solution
of compounds and metal ions, like Na+, K+ and Mg2+. Upon
gradual addition of metal ions, the absorption and fluores-
cence spectra showed gradual decrease in the intensity, how-
ever, the wavelength of absorption and emission spectra
remained unchanged. The fluorescence spectra of compounds
2–6 with univalent ion have been shown in Fig. 2 and with
bivalent ion have been shown in Fig. 3 Univalent ions showed
similar decrease in intensity for each compounds. In the pres-
ence of monovalent Na+ and K+ ions, compounds 4 and 6
showed about 20–22 % decrease whereas compounds 2, 3
and 5 about 8 % decrease in fluorescence. This decrease in
fluorescence in the presence of divalent Mg2+ ion was very
Conclusions
We have developed some new potential fluorescent com-
pounds with high quantum yield. The compounds exhibited
solvatochromic behavior and the fluorescence measurement
studies on these molecules showed emission spectra at longer
wavelength in the region 430–530 nm. TCSPC experiments
revealed a good average lifetime of these molecules under
excited state condition. Compounds retained their stability
and fluorescence property with negligible change in intensity

J Fluoresc
in the presence of ions, like Na+, K+ and Mg2+ under aqueous
conditions and hence are supposed to be efficient and
useful fluorescent reporter groups for drug delivery in
biological systems.
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Acknowledgments The authors would like to thank Dr. Anjan
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them to perform Photophysical studies in his laboratory.
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