Chemiluminescence properties of luminol related quinoxaline analogs: Experimental and DFT based approach to photophysical properties

Article abstract of DOI:10.1016/j.dyepig.2015.02.006

Novel luminol and isoluminol related compounds containing a quinoxaline moiety were synthesized by the reaction of various 1,2-diketones with dimethyl-4,5-diaminophthalate (5) and dimethyl-3,4-diaminophthalate (5′). The UV-Vis absorption and emission spectra of the dyes were studied in solvents of differing polarity and the compounds show solvatofluorism properties. The chemiluminescent properties of the isoluminol and luminol based quinoxaline derivatives were examined and compared with isoluminol and luminol. These compounds produced chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in sodium hydroxide solution. The chemiluminescence intensities of these chemiluminophores were affected by the concentrations of hydrogen peroxide, potassium hexacyanoferrate(III) and sodium hydroxide. These quinoxaline derivatives were found to produce chemiluminescence in the range of 3.2-7.3 and it is 0.57-1.7 times more intensely than isoluminol and luminol, respectively. Density Functional Theory computations have been used for in-depth understanding of structural, molecular, electronic and photophysical parameters of the compounds.

Full text of DOI:10.1016/j.dyepig.2015.02.006

Accepted Manuscript
Chemiluminescence properties of luminol related quinoxaline analogues:
experimental and DFT based approach to photophysical properties
Mininath S. Deshmukh, Nagaiyan Sekar
PII:
S0143-7208(15)00042-X
10.1016/j.dyepig.2015.02.006
DYPI 4660
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 5 October 2014
Revised Date: 12 January 2015
Accepted Date: 6 February 2015
Please cite this article as: Deshmukh MS, Sekar N, Chemiluminescence properties of luminol related
quinoxaline analogues: experimental and DFT based approach to photophysical properties, Dyes and
Pigments (2015), doi: 10.1016/j.dyepig.2015.02.006.
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ACCEPTED MANUSCRIPT
Chemiluminescence properties of luminol related quinoxaline analogues: experimental and DFT
based approach to photophysical properties
Mininath S. Deshmukh, Nagaiyan Sekar*
Tinctorial Chemistry Group, Department of Dyestuff Technology,
Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai - 400 019. (India)
*Corresponding author. Tel.: +91 22 3361 1111/2222, 2707(direct), Fax.: +91 22 3361 1020.
E-mail: n.sekar@ictmumbai.edu.in, nethi.sekar@gmail.com
Abstract:
Novel luminol and isoluminol related compounds containing a quinoxaline moiety were synthesized by
the reaction of various 1,2-diketones with dimethyl-4,5-diaminophthalate (5) and dimethyl-3,4-
diaminophthalate (5’). The UV-Vis absorption and emission spectra of the dyes were studied in solvents
of differing polarity and the compounds show solvatofluorism properties. The chemiluminescent
properties of the isoluminol and luminol based quinoxaline derivatives were examined and compared
with isoluminol and luminol. These compounds produced chemiluminescence by reaction with hydrogen
peroxide in the presence of potassium hexacyanoferrate(III) in sodium hydroxide solution. The
chemiluminescence intensities of these chemiluminophores were affected by the concentrations of
hydrogen peroxide, potassium hexacyanoferrate(III) and sodium hydroxide. These quinoxaline
derivatives were found to produce chemiluminescence in the range of 3.2-7.3 and it is 0.57-1.7 times
more intensely than isoluminol and luminol, respectively. Density Functional Theory computations have
been used for in-depth understanding of structural, molecular, electronic and photophysical parameters
of the compounds.
Keywords:
Luminol-isoluminol;
Chemiluminescence,
Quinoxalines;
Solvatochromism;
Solvatofluorism; TD-DFT.

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1. Introduction
5-Amino-2,3-dihydro-1,4-phthalazine-1,4-dione (luminol) and 6-amino-2,3-dihydro-1,4-phthalazine-
1,4-dione (isoluminol) are the well-known chemiluminescence (CL) compounds, and their derivatives
have been widely used for various biomicroanalyses due to their high selectivity and sensitivity[1]. In
recent years, chemiluminescence has been an attractive detection technique for high-performance liquid
chromatography (HPLC)[2]. A convenient labeling reagent having a luminol moiety has not been
reported due to the fact that the CL intensity is always low and they are not so easily synthetically
accessible. On the other hand, various reagents containing an isoluminol moiety like
isoluminolisothiocyanate
(ILITC)[3],
N-aminobutyl-N-ethylisoluminol
(ABEI)[4,5],
4,5-
diaminophthalhydrazide (DPH)[6] and 6-aminomethylphthalhydrazide (6-AMP)[7] have been widely
used for the microanalyses of biological compounds. The most of these reagents react with the amino
and carboxylic acid groups of the analytes and a new CL labeling reagent which reacts with the other
functional groups is required. The modification of the amino group of luminol with the electron donating
groups such as an alkyl or aryl group causes a decrease in the CL intensity. On the other hand, that of
the isoluminol with an electron donating group causes an increase in the CL intensity[8,9].
The quinoxaline derivatives are also an important class of benzoheterocycles have been widely
used as dyes[10], emitters in organic light emitting diodes, building blocks of organic semiconducting
constituents in thin-film transistors[11], as well as fluorescent sensors and π-conjugating chromophores
in nonlinear optical materials[12–14]. There exists an extensive π-conjugation in quinoxaline based
chromophores and they display a strong emission with relatively low Stokes shifts. But, in the planar
structure, they are prone to stronger intermolecular π-π stacking interactions, which often decrease their
luminescence and increase the tendency to form crystals in the solid state[15] While the stacking
interactions are beneficial for the charge transport properties, crystallinity and decrease in emission yield

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or unwanted red-shift in emission in the solid state pose a problem in the utilization of polyaromatic
hydrocarbon in organic light-emitting diodes[16–18].
In this paper we report the synthesis of π-conjugated luminol and isoluminol based quinoxaline
chromophores with relatively high Stokes shifts. The molecular structures are shown in Scheme 1.
These dyes were obtained by the condensation reaction between dimethyl 4,5-diaminophthalate or
dimethyl 3,4-diaminophthalate and different substituted 1,2-diketone (a-c). Density functional theory
computations [B3LYP/6-31G(d)] were used to study the geometrical and electronic properties of the
1
synthesized molecules. Their structures were confirmed by FT-IR, H NMR and mass spectra. Their
thermal stability, UV-Vis absorption, emission and chemiluminescence characteristics were also studied.
2. Experimental Section
2.1. Materials and Methods
All the commercial reagents and the solvents were purchased from S. D. Fine Chemicals Pvt. Ltd. and
were used without purification. The reaction was monitored by TLC using on 0.25 mm silica gel 60 F254
precoated plates, which were visualized with UV light. Melting points were measured on standard
melting point apparatus from Sunder industrial product Mumbai and are uncorrected. FT-IR spectra
1
were recorded on Jasco 4100 using ATR accessory. H-NMR spectra were recorded on VARIAN Inc.
(USA) 400/500/600-MHz instrument using TMS as an internal standard and CDCl3/DMSO-d6 as the
solvent at room temperature. Mass spectra were recorded on Finnigan mass spectrometer. Simultaneous
DSC-TGA measurements were performed out on SDT Q 600 v8.2 Build 100 model of TA instruments
Waters (India) Pvt. Ltd. The UV-visible absorption and emission, chemiluminescence spectra were
performed on a Perkins-Elmer Lamda 25 and Varian Cary Eclipse at room temperature respectively.
Quantum yields were obtained by using quinine sulfate (0.54 in 0.1 M H2SO4) as reference[19].

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Stock solutions (1x10-3 M) of each chemiluminophore were prepared in N,N-dimethylformamide
(DMF) and these solutions were stable for at least 2 weeks. The solutions of potassium
hexacyanoferrate(III) (2.0-50 mM) and hydrogen peroxide (2.0-50 mM) were prepared in sodium
hydroxide (0.5-3.0M) solution and used within 12 h.
2.2. Computational methods
The ground state (So) geometry of the synthesized compounds were optimized in vacuum using Density
Functional Theory (DFT)[20]. The functional used was B3LYP. The B3LYP method combines Becke’s
three parameter exchange functional (B3)[21] with the nonlocal correlation functional by Lee, Yang,
and Parr (LYP)[22]. The basis set used for all the atoms was 6-31G(d), the latter has been justified in the
literature[23–25] for the current investigation. The vibrational frequencies at the optimized structures
were computed using the same method to verify that the optimized structures correspond to local
minima on the energy surface[26]. The vertical excitation energy and oscillator strengths at ground state
equilibrium geometries were calculated by using TD-DFT at the same hybrid functional and basis
set[27–29]. The low-lying first singlet excited states (S1) of dyes was relaxed using TD-DFT to obtain
its minimum energy geometry. The difference between the energies of the optimized geometries at the
first singlet excited state and the ground state was used in computing the emissions[30,31]. Frequency
computations were also carried out on the optimized geometry of the low-lying vibronically relaxed first
excited state of conformers. All the computations in solvents of different polarities were carried out
using the Polarizable Continuum Model (PCM)[32]. All electronic structure computations were carried
out using the Gaussian 09 program[33].

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2.3. Synthetic strategy
The compounds 7a-7c, 7’a-7’c, 8a-8c and 8’a-8’c contain the donor core 9,10-dihydrophenanthrene/
1,2-diphenylethane/ 1-ethylindoline along with quinoxaline unit and the diester acceptor moiety. They
were synthesized by the conventional condensation of dimethyl 4,5-diaminophthalate (6) and dimethyl
4,3-diaminophthalate (6’) with the different 1,2-diketones (a-c) (Scheme 1).
In the first step, dimethyl-4-nitrophthalate 1 was synthesized by the esterification of 4-
nitrophthalic acid. The compound 1 on reduction by using H2- Pd/C (10%) gave the compound 2, which
on further acetylation using acetic anhydride in toluene yielded the intermediate 3. Nitration of the
intermediate 3 with fuming HNO3 and H2SO4 gave the intermediates 4 and 4’, which on deacetylation in
concentrated H2SO4 gave the intermediates 5 and 5’. Nitro group of the compounds 5 and 5’ was
reduced using H2- Pd/C (10%) to give the compounds 6 and 6’, which on further condensation with the
1,2-diketones (a-c) in glacial acetic acid at reflux temperature gave the compounds 7a-7b and 7’a-7’c.
Finally the compound was refluxed in hydrazine hydrate and triethyl amine in methanol to yield the
desired chemiluminescent compounds 8a-8b and 8’a-8’c respectively.
<< Please insert Scheme 1 >>
2.4. Examination of the chemiluminescent properties
2.4.1. Standard procedure
The chemiluminescence reaction was initiated by the simultaneous automatic injections of 100 µl of
hydrogen peroxide solution and 100 ml of potassium hexacyanoferrate(III) solution dissolved in sodium
hydroxide to the 100 µl portion of a 10 nM solution of each chemiluminophore (Figure 1). The
chemiluminescence intensities were recorded immediately after the injection of oxidizing agent solution.

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The integrated photon counts for 1 min after the injections were explained as the chemiluminescence
intensities.
<< Please insert Figure 1 >>
2.4.2. Optimization of the chemiluminescence conditions
To establish the optimum chemiluminescence conditions the concentrations of the oxidizing reagents
(hydrogen peroxide, potassium hexacyanoferrate(III) and sodium hydroxide) were varied one at a time.
The temporarily settled conditions were DMF solution of chemiluminophore, 10.0 mM hydrogen
peroxide and 10.0 mM potassium hexacyanoferrate(III) prepared in 1.5 M sodium hydroxide.
2.4.3. Fluorescence of the CL Reaction Product
To 200 µl of the 1mM stock solution of chemiluminophores, 50 µl of 10 mM potassium
hexacyanoferrate(III) in 1.5 M NaOH and 50 µl of 10mM hydrogen peroxide were added and allowed to
stand for 1 h at room temperature. The emission maxima, excitation and relative intensities of the
resulting mixtures were recorded in the fluorescence spectrometer.
2.5. Synthesis and characterization
The synthetic scheme for the preparation of luminol-isoluminol based quinoxaline derivatives are shown
in Scheme 1. Dimethyl 4,5-diaminophthalate (6) and dimethyl 4,3-diaminophthalate (6’) were prepared
by the reported procedure[34] from 4-nitrophthalic acid.

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2.5.1. General procedure for the preparation of quinoxalines
Diamino-dimethylphthalate (6 or 6’) 0.5 g (2.2 mmol) and substituted 1,2-diketones (a-c) (2.2 mmol)
were dissolved in acetic acid (5 mL), and the reaction mixture was refluxed for 6 h. The mixture was
poured on the crushed ice and the precipitate obtained was filtered, washed with water and dried to
afford 7a-7c and 7’a-7’c. The product was recrystalized from ethanol.
Dimethyl dibenzo[a,c]phenazine-11,12-dicarboxylate (7a):
o
Yield: 92%; Melting point: 260-262 C; HRMS: Calcd for C24H16N2O4Na [M+Na]+ 419.1008, found
1
419.1008; FAB-MS m/z: 397.47 [M+H]+; H NMR (CDCl3, 600 MHz): δ 9.39 (d, 2H, Ar-H, J = 7.8
Hz), 8.72 (s, 2H, Ar-H), 8.58 (d, 2H, Ar-H, J = 8.4 Hz), 7.86 (t, 2H, Ar-H, J = 7.8 Hz), 7.78 (t, 2H, Ar-
H, J = 7.8 Hz), 4.04 (s, 6H, -OCH3); 13C NMR (CDCl3, 125.8 MHz): δ 167.78, 145.12, 133.63, 131.22,
130.03, 128.75, 127.15, 126.34, 125.87, 124.72, 121.41, 52.07; FT-IR: 1737, 1722 (-C=O ester), 1609,
1569 (C=C, C=N ring stretching), 1271, 1258 (C-O stretching) cm-1.
Dimethyl 2,3-diphenylquinoxaline-6,7-dicarboxylate (7b):
o
Yield: 87%; Melting point: 214-216 C; HRMS: Calcd for C24H19N2O4 [M+H]+ 399.1345, found
1
399.1343; FAB-MS m/z: 399.33 [M+H]+; H NMR (CDCl3, 600 MHz): δ 8.56 (s, 2H, Ar-H), 7.55 (m,
4H, Ar-H), 7.39 (m, 6H, Ar-H), 4.00 (s, 6H, -OCH3); 13C NMR (CDCl3, 125.8 MHz): δ 167.92, 157.63,
144.06, 137.53, 132.43, 131.92, 130.23, 129.41, 128.95, 128.35, 127.21, 51.67; FT-IR: 1740, 1725 (-
C=O ester), 1615, 1589 (C=C, C=N ring stretching), 1261, 1240 (C-O stretching) cm-1.

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Dimethyl 6-ethyl-6H-indolo[2,3-b]quinoxaline-2,3-dicarboxylate (7c):
o
Yield: 90%; Melting point: 184-186 C; HRMS: Calcd for C20H17N3O4Na [M+Na]+ 386.1117, found
1
386.1116; FAB-MS m/z: 364.47 [M+H]+; H NMR (CDCl3, 600 MHz): δ 8.75 (s, 1H, Ar-H), 8.51 (d,
1H, Ar-H, J = 7.8 Hz), 8.46 (s, 1H, Ar-H), 7.78 (t, 1H, Ar-H, J= 9.0 Hz), 7.54 (d, 1H, Ar-H, J = 7.2 Hz),
13
7.78 (t, 1H, Ar-H, J= 7.8 Hz), 4.59 (q, 2H, -NCH2), 4.01 (s, 6H, -OCH3), 1.55 (t, 3H, -CH3); C NMR
(CDCl3, 125.8 MHz): δ 167.83(s), 147.51, 145.12, 142.73(s), 110.13, 132.51(s), 129.83(s), 124.07,
123.45, 121.65, 120.93, 110.17, 52.73, 52.08, 47.35, 14.32; FT-IR: 1745, 1739 (-C=O ester), 1621, 1595
(C=C, C=N ring stretching), 1267, 1242 (C-O stretching) cm-1.
Dimethyl dibenzo[a,c]phenazine-10,11-dicarboxylate (7’a):
o
Yield: 85%; Melting point: 254-256 C; HRMS: Calcd for C24H16N2O4Na [M+Na]+ 419.1008, found
1
419.1013; Mass m/z: 397.40 [M+H]+; H NMR (CDCl3, 400 MHz): δ 9.39 (d, 1H, Ar-H, J = 8.0 Hz),
9.29 (d, 1H, Ar-H, J = 8.0 Hz), 8.55 (t, 2H, Ar-H, J= 7.2 Hz), 8.41 (s, 2H, Ar-H), 7.84 (dd, 2H, Ar-H, J
= 7.2 Hz and J = 8.0 Hz), 7.75 (t, 2H, Ar-H, J = 7.2 Hz and J = 8.0 Hz), 4.25 (s, 3H, -OCH3), 4.05 (s,
13
3H, -OCH3); C NMR (CDCl3, 125.8 MHz): δ 167.34, 162,95, 144.05, 139.12(s), 138.27(s), 136.84,
135.66, 132.72, 131.45 (s), 130.14(s), 129.17(s), 128.55, 128.17(s), 127.69(s), 126.04, 52.07, 51.63; FT-
IR: 1735, 1722 (-C=O ester), 1614, 1578 (C=C, C=N ring stretching), 1280, 1244 (C-O stretching) cm-1.
Dimethyl 2,3-diphenylquinoxaline-5,6-dicarboxylate (7’b):
o
Yield: 80%; Melting point: 202-204 C; HRMS: Calcd for C24H18N2O4Na [M+Na]+ 421.1164, found
1
421.1159; FAB-MS m/z: 399.20 [M+H]+; H NMR (CDCl3, 400 MHz): δ 8.33 (d, 1H, Ar-H, J = 9.2
Hz), 8.23 (d, 1H, Ar-H, J = 9.2 Hz), 7.55 (d, 4H, Ar-H, J = 6.8 Hz), 7.35 (m, 6H, Ar-H), 4.09 (s, 3H, -
OCH3), 4.01 (s, 3H, -OCH3); 13C NMR (CDCl3, 125.8 MHz): δ 167.22, 163,75, 157.13, 148.13, 144.05,

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139.51(s), 137.83, 135.17, 134.02, 133.63, 131.13(s), 131.77(s), 129.44, 128.36(s), 127.61(s), 125.10,
52.33, 51.94; FT-IR: 1741, 1730 (-C=O ester), 1621, 1577 (C=C, C=N ring stretching), 1251, 1232 (C-
O stretching) cm-1.
Dimethyl 6-ethyl-6H-indolo[2,3-b]quinoxaline-3,4-dicarboxylate (7’c):
o
Yield: 85%; Melting point: 176-178 C; HRMS: Calcd for C20H17N3O4Na [M+Na]+ 386.1117, found
386.1114; FAB-MS m/z: 364.42 [M+H]+; 1H NMR (CDCl3, 400 MHz): 8.46 (d, 1H, Ar-H, J = 8.0 Hz),
8.34 (d, 1H, Ar-H, J = 8.8 Hz), 8.18 (d, 1H, Ar-H, J = 8.8 Hz), 7.74 (t, 1H, Ar-H, J = 8.4 Hz), 7.50 (d,
1H, Ar-H, J = 8.0 Hz), 7.78 (t, 1H, Ar-H, J = 8.4 Hz), 4.58 (q, 2H, -NCH2), 4.19 (s, 3H, -OCH3), 4.01 (s,
13
3H, -OCH3), 1.55 (t, 3H, -CH3); C NMR (CDCl3, 125.8 MHz): δ 167.62, 162,95, 148.03, 145.13,
144.36, 137.23, 136.93, 135.67, 134.81, 132.13, 126.61, 123.77, 123.18, 119.74, 119,08, 110.11, 52.67,
52.03, 47.16, 14.28; FT-IR: 1748, 1732 (-C=O ester), 1614, 1571 (C=C, C=N ring stretching), 1254,
1232 (C-O stretching) cm-1.
2.6.2. General procedure for the preparation of phthalazines (8a-8c and 8’a-8’c):
The compounds 7a-7c and 7’a-7’c (1 mmol) and hydrazine hydrates (1.5 ml) were reacted in the
presence of triethyl amine (1.5 ml) in methanol (5 ml) at reflux temperature for 2 h. The product was
precipitated during reflux temperature or at room temperature. The crude product was recrystallized
from N,N’-dimethylacetamide to get the compounds 8a-8c and 8’a-8’c. The low solubility of the
compounds 8a-8c and 8’a-8’c made the 13C-NMR characterization of these substrates not possible. Only
the 1H-NMR, HRMS and IR are recorded.

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12,13-Dihydrodibenzo[a,c]pyridazino[4,5-i]phenazine-11,14-dione (8a):
Yield: 86%; Melting point: > 300 oC; HRMS: Calcd for C22H13N4O2 [M+H]+ 365.1039, found 365.1041;
FAB-MS m/z: 364.40 [M]+; 1H NMR (DMSO-d6, 600 MHz): 11.00 (brs, 2H, -NH), 9.32 (d, 2H, Ar-H, J
= 7.8 Hz), 8.91 (s, 2H, Ar-H), 8.81(d, 2H, Ar-H, J = 8.4 Hz), 7.92 (t, 2H, Ar-H, J = 7.8 Hz), 7.85 (d, 2H,
Ar-H, J = 7.2 Hz); FT-IR: 3014, 2897 (N-H amide), 1665, 1625 (-C=O amide), 1602, 1536 (C=C, C=N
ring stretching), 1315, 1268 (C-N stretching) cm-1.
2,3-Diphenyl-7,8-dihydropyridazino[4,5-g]quinoxaline-6,9-dione (8b):
Yield: 84%; Melting point: > 300 oC; HRMS: Calcd for C22H15N4O2 [M+H]+ 367.1195, found 367.1149;
FAB-MS m/z: 367.27 [M+H]+; 1H NMR (DMSO-d6, 600 MHz): 11.76 (s, 2H, -NH), 8.83 (s, 1H, Ar-H),
8.64 (s, 1H, Ar-H), 7.53 (d, 4H, Ar-H, J = 7.8 Hz), 7.43 (t, 2H, Ar-H, J = 7.2 Hz), 7.38 (t, 4H, Ar-H, J =
7.2 Hz); FT-IR: 3014, 2893 (N-H amide), 1661, 1626 (-C=O amide), 1600, 1595 (C=C, C=N ring
stretching), 1366, 1340 (C-N stretching) cm-1.
7-Ethyl-2,3-dihydro-1H-indolo[2,3-b]pyridazino[4,5-g]quinoxaline-1,4(7H)-dione (8c):
o
Yield: 77%; Melting point: > 300 C; HRMS: Calcd for C18H15N5O2 [M+2H]+ 333.1226, found
1
333.2879; FAB-MS m/z: 332.23 [M+H]+; H NMR (DMSO-d6, 600 MHz): 11.56 (brs, 2H, -NH), 8.88
(s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 8.42 (d, 1H, Ar-H, J = 7.8 Hz), 7.83 (s, 2H, Ar-H), 7.44 (t, 1H, Ar-H,
J = 5.4 Hz), 4.54 (q, 2H, -NCH2), 1.43 (t, 3H, -CH3); FT-IR: 3029, 2903 (N-H amide), 1660 (-C=O
amide), 1613, 1578 (C=C, C=N ring stretching), 1315, 1241 (C-N stretching) cm-1.

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2,3-Dihydrodibenzo[a,c]pyridazino[4,5-h]phenazine-1,4-dione (8’a):
o
Yield: 80%; Melting point: > 300 C; HRMS: Calcd for C24H15N5O2 [M+CH3CN]+ 405.1226, found
405.1351; FAB-MS m/z: 364.40 [M+H]+; 1H NMR (DMSO-d6, 600 MHz): 13.31 (s, 1H, -NH), 12.54 (s,
1H, -NH), 9.58 (d, 2H, Ar-H, J = 7.8 Hz), 8.56 (d, 1H, Ar-H, J = 7.2 Hz), 8.24 (d, 1H, Ar-H, J = 7.2
Hz), 7.91(d, 2H, Ar-H, J = 7.8 Hz), 7.64 (t, 2H, Ar-H, J = 7.2 Hz), 7.35 (d, 2H, Ar-H, J = 7.8 Hz); FT-
IR: 3160, 3009 (N-H amide), 1670, 1649 (-C=O amide), 1602, 1567 (C=C, C=N ring stretching), 1356,
1283 (C-N stretching) cm-1.
7-Hydroxy-2,3-diphenyl-8,9-dihydropyridazino[4,5-f]quinoxalin-10(7H)-one (8’b):
Yield: 78%; Melting point: > 300 oC; HRMS: Calcd for C22H15N4O2 [M+H]+ 367.1195, found 367.1196;
1
FAB-MS m/z: 389.27 [M+Na]+; H NMR (DMSO-d6, 600 MHz): 12.88 (s, 1H, -NH), 12.42 (s, 1H, -
NH), 8.56 (dd, 2H, Ar-H, J = 9.0 Hz and J = 9.6 Hz), 7.55 (t, 4H, Ar-H, J = 6.0 Hz), 7.50 (t, 2H, Ar-H,
J = 7.2 Hz), 7.46 (t, 2H, Ar-H, J = 6.6 Hz), 7.41 (t, 2H, Ar-H, J = 7.8 Hz); FT-IR: 3055, 2903 (N-H
amide), 1665 (-C=O amide), 1602, 1564 (C=C, C=N ring stretching), 1354, 1294 (C-N stretching) cm-1.
12-Ethyl-2,3-dihydro-1H-indolo[3,2-b]pyridazino[4,5-f]quinoxaline-1,4(12H)-dione (8’c):
Yield: 82%; Melting point: > 300 oC; HRMS: Calcd for C18H14N5O2 [M+H]+ 332.1147, found 332.1145;
FAB-MS m/z: 332.20 [M+H]+; 1H NMR (DMSO-d6, 600 MHz): 13.29 (s, 1H, -NH), 12.28 (s, 1H, -NH),
8.58 (d, 1H, Ar-H, J = 7.8 Hz), 8.50 (s, 2H, Ar-H), 7.95 (d, 1H, Ar-H, J = 7.8 Hz), 7.90 (t, 1H, Ar-H, J =
7.8 Hz), 7.52 (t, 1H, Ar-H, J = 7.8 Hz), 4.63 (q, 2H, -NCH2), 1.45 (t, 3H, -CH3); FT-IR: 3042, 2897 (N-
H amide), 1654, 1617 (-C=O amide), 1580, 1560 (C=C, C=N ring stretching), 1330, 1295 (C-N
stretching) cm-1.

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3. Results and discussion:
3.1. Photo-physical properties
The UV-Vis absorption and emission spectra of the phthalate and luminol-isoluminol related
quinoxaline compounds in the solvents of varying polarities are reported in Tables S1-S7. The phthalate
quinoxaline derivatives have the solubility mostly in all the organic solvents but luminol-isoluminol
quinoxaline have the solubility only in highly polar solvents like DMF and DMSO. The absorption
spectra of the compounds in all the studied solvents are almost nearly the same; their absorption
property is independent of the solvent polarity (Figure S1-S12). The rigidity in the structure exhibited a
strong red-shifted absorption shown in Figure 2, Tables 1, S1-S7. The compounds 7a, 7c, 7’a, 7’c and
8a, 8c, 8’a, 8’c have four absorption maxima. Among these four transitions, first three are higher energy
absorptions between 270 nm-380 nm probably due to the π-π* and n- π* transition while the lower
energy band between 390 nm and 410 nm attributed to the intramolecular charge transfer transition
(ICT) between the donor group and the acceptor moiety. Also, all these compounds exhibit highly
intense first absorption peak at 270-300 nm due to the aryl core[35], while the absorption spectra of the
compounds 7b 7’b and 8b, 8’b contain two absorption maxima. Among these two, the first one is a high
energy absorptions between 270 nm-320 nm probably due to the π-π* transition while the low energy
band between 320 nm and 370 nm is attributed to the intramolecular charge transfer transition (ICT).
The emission spectra of the compounds 7a, 7c, 7’a, 7’c and 8a, 8c, 8’a, 8’c exhibited a strong
solvent effect as compared to the compounds 7b, 7’b and 8b, 8’b. In the protonated solvents all the
compounds show emission maxima at longer wavelengths than in the other solvents (Tables S1-S7,
Figures S1-S12). In the polar solvents like methanol, DMF the emissive S1 state with intramolecular
charge transfer (ICT) character is strongly solvated and its energy is dramatically lowered. The rigidity
in the molecules 7a, 7c, 7’a, 7’c and 8a, 8c, 8’a, 8’c leads to a red shift (lower energy) in the emission

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(Figure 3). The extent of red shift in emission of the above molecules is more as compared to the red
shift in the corresponding absorption values which may be attributed to more energy losses due to
dissipation of the vibrational energy during the decay and is influenced by the interaction between the
fluorophore and the solvent molecules around the excited dipole, hydrogen bonding and the formation of
charge complexes.
<< Please insert Figure 2 >>
<< Please insert Figure 3>>
<< Please insert Table 1>>
3.2. Quantum yield of Compounds:
The fluorescence quantum yields of the synthesized quinoxaline compounds were determined in
different solvents and tabulated in Tables 1, S1-S7. The fluorescence quantum yields of the compounds
are mostly depending on both the nature of the substituent and the solvent polarity. Here, it has been
observed that the compounds 7a-7c, 7’a-7’b and compounds 8a-8c, 8’a-8’c enhances the fluorescence
quantum yields in the polar protic (ethanol) and polar aprotic (DMF) solvent respectively. The dyes
showed higher quantum efficiencies in the non-polar solvents while lower quantum yields in the polar
solvents. The highest quantum yield of the compounds 7a-7c, 7’a-7’c and 8a-8c, 8’a-8’c was observed
in ethanol and DMF and the values in the increasing order are: 7’b (0.04) < 7’a (0.07) = 7’a (0.07) < 7a
(0.12) < 7’c (0.13) < 7c (0.15) and 7’b (0.04) < 7c (0.08) = 7’c (0.08) < 7a (0.09) < 8a(0.10) = 8’a(0.10).
The quantum yield values were the lowest in DCM in the decreasing order: 7’c (0.11) > 8’a (0.10) > 7a
(0.09) = 7c (0.09) > 8c (0.07) > 8a (0.06) = 8’c (0.06) > 7’a (0.05) = 8b (0.05) > 7b (0.04) > 7’b (0.03)
= 8’b (0.03). These compounds showed comparatively less emission intensity in acetonitrile, DMSO and
DMF showing a clear positive solvatokinetic behavior suggesting that a highly polar excited-state
population charge transfer state and a non-radiative decay was prominent in these compounds[30].

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3.3. Chemiluminescence Study
The chemiluminescence properties of isoluminol-luminol type of derivatives 8a-8c and 8’a-8’c were
examined in order to evaluate their use as highly chemiluminescent probes. These six compounds, like
luminol and isoluminol produce chemiluminescence by the reaction with hydrogen peroxide in the
presence of potassium hexacyanoferrate(III) in alkaline medium. The concentrations of the three
oxidizing reagent systems have a major influence on the chemiluminescence intensity (Figure 4); 2.5-20
mM hydrogen peroxide, 2.5-20 mM potassium hexacyanoferrate(III) and 0.5-2.0 M sodium hydroxide
give the maximum intensity. After the addition of hydrogen peroxide and potassium
hexacyanoferrate(III) the chemiluminescence intensity reaches a maximum in a few seconds and then
almost disappears within one minute.
Under these optimum reaction conditions, the chemiluminescence intensities of the six compounds are
determined (Table 2). The generation of the chemiluminescence of the compounds 8a-8c and 8’a-8’c
are found approximately 3.2-7.3 times larger than that of the isoluminol, and the intensities
corresponded to 0.57-1.7 times of that obtained with the luminol. The time dependence of the
chemiluminescence reaction of the synthesized compounds and luminol-isoluminol is illustrated in
Figure 5. The generation of chemiluminescence is initiated by the addition of hydrogen peroxide and
potassium hexacyanoferrate(III) in alkaline solutions to the stock solution. The chemiluminescence
intensity was maximum at <1.5 s after the addition of the oxidizing reagents, and then decreased rapidly.
In the chemiluminescence of luminol and isoluminol, the 3-aminophthalate and 4-aminophthalate ion
produced during the oxidizing reaction have been shown to be the light emitter. Therefore, the newly
synthesized luminol-isoluminol type compounds, and the corresponding dicarboxylate ions (Figure 1)
could be expected to be the light emitting species. The excitation and emission maxima of the

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fluorescence and the relative intensity of the chemiluminescent species on the completion of the
chemiluminescence reaction are measured, and compared with the conventional luminol analogues
(Table 2). The fluorescence intensities of the dicarboxylate ions of the quinoxaline derivatives are 1.1-
12.5 times larger than that of 3-aminophthalate anion of the luminol. The result proposes that the
efficiency of the chemiluminescencent compound is mostly dependent on the fluorescence intensities of
the light-emitting species produced during the reaction. Otherwise, it is known that the
chemiluminescence spectra of the isoluminol and luminol type compounds are almost identical to the
emission spectra of the corresponding phthalate ion[36] (Figure 1). Thus, the compounds 8a, 8c and
8’a-8’c are expected to generate chemiluminescence at longer wavelengths as compared to the
isoluminol and luminol respectively.
<< Please insert Figure 4 >>
<< Please insert Figure 5 >>
<< Please insert Table 2 >>
3.4. Geometry optimization
The ground state geometries of the compounds 7a, 7c, 7’a, 7’c, 8a, 8c, 8’a and 8’c were having planar
arrangement of the condense ortho-diamino with diketone due to their rigidity. While, the compounds
7b, 7’b, 8b and 8’b both the phenyl ring on quinoxaline is twisted. In the phthalate compounds 7a-7c
and 7’a-7’c the ester group have small twist with the quinoxaline.
As a representative example, the structural view of the compound 7b in the ground and the excited state
in DMF is presented in Table 3, Figure 6 and the structures of the remaining compounds are
summarized in Tables S8-S10, Figures S13-S23. In the dye 7b major bond lengthening was observed
between the bonds C1-C2, C3-N4, C5-C6, C9-O10, C20-C25, C22-C21, C29-N19 by 0.032, 0.018, 0.003, 0.007,

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0.012, 0.012, 0.05 Å and the bond length shortened for the bonds C2-C6, C4-C5, O11-C14, C22-C26, C23-
C24, C26-C29 by 0.012, 0.003, 0.002, 0.02, 0.007, 0.039 Å. Such a lengthening and shortening of the
bonds are due to the effective charge delocalization in the molecules. Also, the excited state twist
dihedral angle of both the phenyl ring with quinoxaline unit was reduced to maintain the planarity.
<< Please insert Figure 6 >>
<< Please insert Table 3 >>
Twisted geometry in the compounds 7b, 7’b, 8b and 8’b
The ground state optimized geometry of the dyes 7b, 7’b, 8b and 8’b in DMF has a dihedral
angle between both the phenyl ring and the quinoxaline C23-C22-C26-C29/C27-C28-C29-C26 40o-44o. The
ester groups of the compounds 7a-7c and 7’a-7’c have a larger dihedral angle with the quinoxaline unit
than the fused phthalazine based quinoxaline compounds 8a-8c and 8’a-8’c. In the excited state of the
compounds 7b, 7’b, 8b and 8’b in DMF both the phenyl ring was twisted to a reduced dihedral angle
between C23-C22-C26-C29/C27-C28-C29-C26 up to 31o-38o. Similarly the terminal ester and 1,2-
dihydropyridazine-3,6-dione are also twisted to a reduced dihedral angle amounting to reaching a
planarity in the excited state (Table 4).
<< Please insert Table 4 >>
The Mulliken charge distribution in the ground and excited state (DMF solvent) of all the compounds
are summarized in Tables S11-S14, Figures S24-S34. In the excited state of the compound 7a, net
positive charge on the atom C26 and C29 increases from 0.257 to 0.263. Also the net negative charge on
N18/19 and C3 atom increases from -0.606 to -0.608 and -0.220 to -0.224 (in au) (Figure 7),The above
observations are suggestive of charge delocalization in the compounds 7a-7c, 7’a-7’c and 8a-8c, 8’a-

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8’c from different donor 9,10-dihydrophenanthrene/ 1,2-diphenylethane/ 1-ethylindoline along with the
quinoxaline unit to the diester and the 2,3-dihydrophthalazine-1,4-dione acceptor moiety respectively.
These atoms are also engaged in bond lengthening and shortening at the excited state. The structures
indicating charge distribution on the dyes were visualized using GaussView 5.0 software[37].
<< Please insert Figure 7 >>
3.5. Electronic vertical excitations (TD-DFT)
The electronic vertical excitations were calculated using TD-B3LYP/6-31G(d) method. The
experimental absorption wavelengths and the computed vertical excitation spectra associated with their
oscillator strengths, composition, and their corresponding assignments of the compounds 7a-7c, 7’a-7’c,
8a-8c and 8’a-8’c are summarized in Tables 1, S1-S7. The absorption band occurring at a lower energy
is due to the intramolecular charge transfer (ICT) and it is the characteristic of the donor-π-acceptor
push-pull dyes. These ICT bands for the compounds 7a-7c, 7’a-7’c and 8a-8c, 8’a-8’c were mainly due
to the electronic transitions from the highest occupied molecular orbital (HOMO) and HOMO-1 to the
lowest unoccupied molecular orbital (LUMO) respectively. While remaining transition assign from π-π*
and n- π* is summarized in Table S1-S7. The experimental absorption and computed vertical excitation
of the all compounds are independent on the solvent polarity, while Stokes shift increases progressively
with the increase in solvent polarity. These observations are in accordance with the higher stabilization
in increasingly polar protic solvents. Emission values of the compound 7a were computed in all solvents
and compounds 7b-7c, 7’a-7’c, 8a-8c and 8’a-8’c in DMF. The computational emission results obtained
by using TD-B3LYP/6-31G(d) were not able to reproduce the solvatofluorism behavior and shown
almost nearly same value of the emission energy.

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As a representative example dimethylphthalate quinoxaline 7a, experimental absorption and it’s
computed vertical excitation values in DMF are 410, 361, 349, 261 nm and 417, 347, 344, 300 nm due
to the HOMO→LUMO, HOMO-1→LUMO, HOMO-2→LUMO, HOMO→LUMO+1 respectively.
There is a significant difference (~ 14 nm) and % deviation between experimental absorption and
vertical excitation is between 1.294-3.96% in studied solvents. The largest wavelength difference
between experimental absorption maxima obtained from ICT and computed vertical excitation was 9 nm
(DMF), 13 nm (DCM), 8 nm (acetone), 16 nm (methanol), 19 nm (DMF) and 24 nm (acetone) for
compounds 7a, 7b, 7c, 7’a, 7’b and 7’c respectively.
The first excited state of the compounds 7a in all the solvents and 7b-7c, 7’a-7’c, 8a-8c, 8’a-8’c in DMF
were optimized to calculate the fluorescence. The experimentally obtained emission spectral data and
the emission computed from TD-B3LYP/6-31G(d) computations are shown in Table 5. The
computational results obtained by using TD-B3LYP/6-31G(d) were not able to reproduce the
solvatofluorism behavior of the dye 7a and showed almost nearly same value of emission energy. The
difference between the energies of the optimized geometries at the first singlet excited state and the
ground state are used to calculate emissions are summarized in Table S15-S16. The experimental
emission wavelength and emission computed by TD-B3LYP/6-31G(d) showed a largest difference of 58
nm in methanol and showed a closest difference of 13 nm in acetone. Similarly, difference between the
experimental emission wavelength and the emission computed by TD-B3LYP/6-31G(d) in DMF of the
compounds 7b, 7c, 7’a-7’c, 8a-8c and 8’a-8’c summarized in (Table 6).
<< Please insert Table 5 >>
<< Please insert Table 6 >>

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As a representative example phthalazine based quinoxaline 8a, experimental absorption and it’s
computed vertical excitation values in DMF are 411, 394, 303, 267 nm and 408, 389, 316, 273 nm due
to the HOMO-1→LUMO, HOMO-2→LUMO, HOMO→LUMO+1, HOMO-2→LUMO+1 respectively.
There is a significant difference (~ 3 nm) and % deviation between experimental absorption and vertical
excitation in DMF is 0.8 %. The wavelength difference between experimental absorption maxima
obtained from ICT and the computed vertical excitation of the compounds 8b, 8c, 8’a, 8’b and 8’c in
DMF was 14, 32, 4, 9 and 2 nm respectively (Tables S1-S7).
The compounds 8a-8c and 8’a-8’c were optimized in the first excited state to calculate the fluorescence
in DMF solvent. The experimentally obtained emission spectral data and the emission computed from
TD-B3LYP/6-31G(d) computations are shown in Table 6. The % deviation between the experimental
emission wavelength and the emission computed by TD-B3LYP/6-31G(d) of the compounds 8a, 8b, 8c,
8’a, 8’b and 8’c in DMF is 4.4, 10.2, 7.4, 3.5, 6.5, 4.5, 3.6, 3.6, 11.4, 1.6 and 8.0 respectively.
3.6. Frontier molecular orbitals
The different frontier molecular orbitals were studied to understand the electronic transition and charge
delocalization within the molecules. The comparative increase and decrease in the energy of the
occupied (HOMO’s) and virtual orbitals (LUMO’s) gives a qualitative idea of the excitation properties
and the ability of the hole or electron injection[10]. The first allowed and the strongest electron
transitions usually correspond almost exclusively to the ICT of an electron from HOMO → LUMO and
HOMO-1 → LUMO for dimethylphthalate and phthalazine quinoxaline respectively (Table S1-S7) and
shows the energies of different molecular orbitals involved in the electronic transitions of these
compounds in different solvents. In the case of all the synthesized quinoxaline compounds 7a-7c, 7’a-
7’c and 8a-8c, 8’a-8’c energy gap of the HOMO → LUMO and HOMO-1 → LUMO orbital remained

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same as the solvent polarity was increased or decreased (Tables S17-S25). The HOMO and LUMO as
well as HOMO-1 and LUMO energy level of the compounds 7a-7’a, 7b-7’b, 7c-7’c and 8a-8’a, 8b-8’b,
8c-8’c in DMF it remained same, and it may be due to the very similar oxidation and reduction potential
(Figure 8). This is understandable because of these structural isomer having same oxidation and
reduction site[38].
<< Please insert Figure 8 >>
Molecular orbital diagram of the compounds 7a-7c, 7’a-7’c and 8a-8c, 8’a-8’c are shown in
Tables S26-S27. From the pictorial diagram the compounds 7a-7c, 7’a-7’c and 8a-8c, 8’a-8’c was
found that HOMO → LUMO and HOMO-1 → LUMO orbitals are fully delocalized throughout the
molecule respectively. The electron densities of the compounds 7a-7c, 7’a-7’c and 8a-8c, 8’a-8’c in the
HOMO and HOMO-1 were slightly more located on the donor moiety 9,10-dihydrophenanthrene/ 1,2-
diphenylethane/ 1-ethylindoline along with quinoxaline unit and the electron densities on the LUMOs
were found localized on the acceptor end. The excitation from HOMO and HOMO-1 to LUMO mostly
consists of charge transfer from the donor core to the acceptor end. Figure 9 contains FMO of dye 7a in
the ground state, which shows that the electron densities are located on the donor 9,10-
dihydrophenanthrene and quinoxaline moiety in the HOMO and these electron densities were shifted
towards the acceptor moiety in the LUMO. Similar observation is seen in all the compounds (Tables
S26-S27).
<< Please insert Figure 9 >>
3.7. Thermal Stability
The dye molecules should form compact aggregates due to the strong intermolecular interactions so that
they acquire high thermal stability[39]. In order to assess the thermal properties of the compounds,

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thermal stability studies have been carried out using thermal gravimetric techniques (TGA) in the
o
o
temperature range 40-600 C under nitrogen gas at a heating rate of 10 C min-1. The TGA results
o
indicated that the synthesized quinoxaline derivatives are stable up to 270 C. TGA revealed the onset
decomposition temperature (Td) of compounds 7a, 7b, 7c, 7’a, 7’b, 7’c, 8a, 8b, 8c, 8’a, 8’b and 8’c are
294 oC (99%), 275 oC (98%), 271 oC (96%), 298 oC (99%), 270 oC (98%), 273 oC (98%), 383 oC (92%),
o
o
o
o
o
350 C (91%), 370 C (82%), 385 C (91%), 300 C (97%) and 338 C (97%) respectively. In general
o
o
the backbones of these quinoxaline derivatives are stable up to 270 C and above 270 C the
thermogravimetric curve of these showed a major loss in weight. The TGA results showed that
quinoxaline derivatives have good thermal stability. Compounds 7a, 7b, 7’a, 7’b, 7’c, 8’b and 8’c
o
o
o
o
o
o
showed a sharp decomposition after temperature 294 C, 275 C, 298 C, 270 C, 273 C, 300 C and
o
o
338 C respectively and completely decomposed beyond 400 C. However, the compounds 7c, 8a, 8b,
o
8c and 8’a showed sluggish decomposition nature and completely decomposed beyond 600 C. The
observations are summarized in Figure 10.
<< Please insert Figure 10 >>
4. Conclusion
In this paper we have reported new chemiluminescent analogues containing an inbuilt quinoxaline
residue and their photophysical and chemiluminescence properties by reaction with hydrogen peroxide
in the presence of potassium hexacyanoferrate (III) in an alkaline medium. The compounds 8a, 8b, 8c,
8’a, 8’b and 8’c can be used as promising candidate for chemiluminescent probes. Photophysical
properties of the synthesized compounds 7a-7c, 7’a-7’c, 8a-8c and 8’a-8’c were supported by DFT and
it was observed that computational results are good agreement with the theoretical observations. The
1
13
synthesized compounds were confirmed by IR, H-NMR, C-NMR and mass spectral analysis. The

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thermal stability was determined by TGA analysis and it was found that the compounds are thermally
stable
.
Acknowledgements
The authors are greatly thankful to TIFR, SAIF-I.I.T. Mumbai for recording the 1H-NMR, 13C-NMR and
Mass spectra. One of the authors Mininath S. Deshmukh is grateful to CSIR for financial support.
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