Effects of homogeneous media, binary mixtures and microheterogeneous media on the fluorescence and fluorescence probe properties of some benzo[b][1,8]naphthyridiens with HSA and BSA

Article abstract of DOI:10.1002/bio.1364

A rapid and efficient method for the synthesis of various poly-substituted benzo[b][1,8]naphthyridines in high yield has been developed via the Friedlaender condensation of 2-aminoquinoline-3-carbaldehyde 1 with various alicyclic ketones in a base catalys

Full text of DOI:10.1002/bio.1364

Research article
Received: 26 May 2011,
Revised: 30 July 2011,
Accepted: 1 September 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.1364
Effects of homogeneous media, binary
mixtures and microheterogeneous media on
the fluorescence and fluorescence probe
properties of some benzo[b][1,8]
naphthyridiens with HSA and BSA
Deepak P. Shelar, Ramhari V. Rote, Sandeep R. Patil and
Madhukar N. Jachak*
ABSTRACT: A rapid and efficient method for the synthesis of various poly-substituted benzo[b][1,8]naphthyridines in high
yield has been developed via the Friedländer condensation of 2-aminoquinoline-3-carbaldehyde 1 with various alicyclic
ketones in a base catalyst (aq. potassium hydroxide). A series of benzo[b][1,8]naphthyridines branched with various side-
chains and substituents were prepared with the aim of being investigated as a fluorescent agents. Electronic absorption
and fluorescence properties of some representative benzonaphthyridines (3d, 5b and 21f) in homogeneous organic solvents,
dioxane–water binary mixtures and in the microheterogeneous media (sodium dodecyl sulphate (SDS), cetyl trimethyl
ammonium bromide (CTAB) and Triton-X100 micelles) have been examined. A linear correlation between solvent polarity
and fluorescence properties was observed. Further, the interaction of these benzonaphthyridines (3d, 5b and 21f) with human
serum albumin (HSA) and bovine serum albumin (BSA) in phosphate buffer have been examined by UV-vis absorption and
fluorescence spectroscopy. The fluorescence intensity of 3d, 5b and 21f increases with the increasing HSA and BSA concentra-
tion. These benzonaphthyridines also quench the 345 nm fluorescence of BSA in phosphate buffer (lex 280 nm). These
compounds have potential for use as neutral and hydrophobic fluorescence probes for examining the microenvironments
in proteins, polymers, micelles, etc. Copyright © 2011 John Wiley & Sons, Ltd.
The following supporting information may be found in the online version of this article.
Keywords: benzo[b][1,8]naphthyridines; microheterogeneous media; human serum albumin (HSA); bovine serum albumin (BSA);
quenching
fluorescence and circular dichroism spectroscopy. Based on such
studies, information on the binding process of many exogenous
Introduction
Every protein has a unique function in metabolism. Proteins are
important for the structural and functional organization of cells
and various cell organelles. Albumins are major proteins in blood
plasma and functions as a transport protein for many compounds,
such as fatty acids, hormones, bilirubin and drugs. Many drugs
and other bioactive small molecules can bind reversibly to
albumins, whereby the latter serves as a carrier. Furthermore,
albumins are the principal biomacromolecules that are involved
in the maintenance of colloid-blood pressure and are implicated
in the facilitated transfer of many ligands across organ–circulatory
interfaces such as in the liver, intestine, kidney and brain (1).
Serum albumin is the most abundant transport protein of all
the proteins in blood plasma, accounting for approximately
60% of the total serum protein content, it has many physiological
and pharmacological functions and have been extensively
studied (2–6). Serum albumin is involved in binding and carriage
of an array of biologically important compounds (exogenous
ligands), such as fatty acids, bilirubin, bile salts and lecithin, and
this binding mechanism was examined using absorption,
ligands has been reported at the molecular level (7–11). However,
major interest in albumins is a result of their strong drug-binding
capacity; it served as model protein for a large variety of biochem-
ical and biophysical studies and a large volume of literature is avail-
able. Numerous experiments are performed for characterizing the
binding capacity and sites of albumins (12,13). Structural aspects
and properties of this transport protein are well explored. The
primary structure is made up of 580 amino acid residues and
secondary structure is constituted of 67% of helix of six turns and
17 disulphide bridges. The tertiary structure is composed of three
domains I, II and III; each domain is constituted by two subdomains,
named as IA, IB, IIA, IIB, IIIA and IIIB (2,4,5). The principal function of
* Correspondence to: M. N. Jachak, Organic Chemistry Research Centre,
Department of Chemistry, KTHM College, Gangapur Road, Nashik 422002,
MS, India. E-mail: mnjachak@hotmail.com
Organic Chemistry Research Centre, Department of Chemistry, KTHM
College, Gangapur Road, Nashik, 422002, MS, India
Luminescence 2011
Copyright © 2011 John Wiley & Sons, Ltd.

D. P. Shelar et al.
serum albumin is to transport a wide variety of fatty acids and
metabolites (4) via the main binding regions located in subdomains
IIA (site 1) and IIIA (site 2) (4,14). Bovine and human serum
albumins (BSA and HSA) display approximately 80% sequence
homology and a repeating pattern of disulphides, which are
strictly conserved.
Experimental
General
Human serum albumin (HSA) and bovine serum albumin (BSA) were
purchased from Hi-Media Laboratories Pvt. Ltd (Mumbai, India). Surfac-
tants [cetyl trimethyl ammonium bromide (CTAB), sodium dodecyl
sulphate (SDS) and Triton-X100 for micelle preparation] and quinine
sulphate [for determination of Φf] were purchsaed from Hi-Media Labo-
ratories and Research-Lab Fine Chem Industries (Mumbai, India), respec-
tively. All other chemicals, reagents and solvents [such as acetonitrile,
dimethylformamide (DMF), dioxane, ethanol, ethyl acetate, n-hexane,
pet-ether methanol and tetrahydrofuran (THF)], used in spectroscopic
and other studies, were obtained from LOBA Chemie. Pvt. Ltd (Mumbai,
India), Spectrochem (Mumbai, India) and E. Merck (India). Deionized, double-
distilled water (Millipore) was used for preparing the micelle solutions. All
AR-grade organic solvents were dried and freshly distilled prior to use. The
UV-grade solvents were used for spectral studies. Melting points were
determined on a Gallenkamp melting-point apparatus, Mod. MFB595, in
open capillary tubes and were uncorrected. Fourier transform infrared
(FTIR) spectra in KBr disk were measured on a Shimadzu FTIR-408 spectro-
photometer. 1H-NMR and 13C-NMR spectra were recorded on a Varian
XL-300 MHz spectrometer, using tetramethylsilane (TMS) as the internal
standard, and the solvents were deuterio-chloroform (CDCl3) and deuterio-
dimethylsulphoxide (DMSO-d6). Chemical shifts were reported in ppm from
an internal tetramethylsilane standard and were given in d-units. Mass
spectra were recorded on a Shimadzu GC–MS QP 2010A mass spectrometer
with an ionization potential of 70 eV. Elemental analyses were performed on
a Hosli CH-analyzer and within Æ 0.3 of the theoretical percentage. For the
microwave irradiation, a conventional (unmodified) domestic BPL (BMO
700T) microwave oven was used. The absorption spectra were measured
using a Shimadzu UV-1601 UV-VIS spectrophotometer. The fluorescence
spectra were recorded on a RF-5301 PC spectrofluorophotometer by
exciting the samples at their absorption maximum (labs. max.). Compounds
for UV and fluorescence measurements were dissolved in DMF, UV and
fluorescence scan were recorded in the range 200–600 nm. The Фf relative
to quinine sulphate in 1.0 Â 10–3 mol/L H2SO4 (Фf = 0.57) was measured at
room temperature by a standard literature procedure (65–67). Both samples
and standard were excited at the same excitation wavelength and the
optical density (OD) of the standard and the sample were adjusted to be
nearly equal. For all electronic spectroscopic studies (such as absorption,
fluorescence excitation and emission) 1.0 Â 10–3 mol/L solutions of the
compounds were used. All reactions were monitored by thin-layer chroma-
tography, carried out on 0.2 mm silica gel 60 F254 (Merck) plates, using UV
light (250 and 400 nm) and fluorescence light (400 and 600 nm) for detection.
For fluorescence quenching studies, the required amount of HSA and BSA so-
lution (in phosphate buffer) and the required amount of benzo[b][1,8]
naphthyridines solution in dimethyl sulphoxide (DMSO) (1.0 Â 10–4 mol/L)
were taken in a 5 mL volumetric flask. Since the benzo[b][1,8]naphthyridines
were sparingly soluble in water, their stock solutions were prepared in DMSO,
which was used as a solvent for interaction studies of serum albumins (37).
Human and bovine serum albumins (HSA and BSA) are prob-
ably the most-studied serum albumin proteins. The protein
HSA has the ability to bind several ligands, including small
aromatic and heterocyclic carboxylic acids, such as non-steroidal
antiflammatory drugs (7). Bovine serum albumin (BSA) has been
one of the most extensively studied proteins of this group.
Various methods are reported to determine bovine serum
albumin, such as resonance light scattering techniques (15–17),
chemiluminescence (18), the Bradford assay method (19), circu-
lar dichroism, fluorescence spectroscopy (20), Fourier transform
infrared spectroscopy (21), ultraviolet absorption spectroscopy,
differential scanning calorimetry (22), etc. Because proteins bear
tryptophan, tyrosine and phenylalanine residues, which have
intrinsic fluorescence, it is seen that fluorescence spectroscopy
could serve as an effective and cheaper method for such studies
(23,24).
Fluorescence probe techniques represent the most important
area of fluorescence spectroscopy to study biologically important
systems and are rapidly gaining popularity in the study of bio-
logical microheterogeneous systems, due to their non-invasive
nature and the large amount of information gleaned thereby
(25,26). Probes that show massive spectral changes on being
bound to such systems are being designed and developed, and
their spectral responses are then exploited to gain information
about the structure and dynamics of the system under study.
Probes such as ANS (27–31) show polarity-dependent spectral
characteristics and are being widely used as fluorescent labels
for studying proteins, vesicles, micelles, mixed micelles and
similar biological or biomimetic micro-heterogeneous systems.
Fluorescence probe techniques are one of the most powerful
methodologies, yielding structural and dynamical informa-
tion concerning the fluorophore environment (8,25,32–37).
Fluorescence spectroscopy and probe molecules that exhibit
solvatochromic fluorescence have been particularly useful for this
purpose (8,31,38–41).
We have recently reported the synthesis of fluorescent benzo
[b][1,8]naphthyridine-3-carbonitrile using 2-aminoquinoline-3-
carbaldehyde (o-aminoaldehyde) 1 (42) and also reported that
certain banzonaphthyridines are capable of solvatochromic
fluorescence emission and these are used as a fluorescence
probe for studying the microenvironment of organized assem-
blies, such as bovine serum albumin (BSA) (43). Herein, we report
a useful and simple approach toward the synthesis of various
benzo[b][1,8]naphthyridines via Friedländer condensation of 1
with amides, benzylcyanide and various cyclic ketones in
different reactions and conditions are discussed. Some repre-
sentative benzonaphthyridines (3d, 5b and 21f) are investi-
gated for their absorption and fluorescence properties in
homogeneous media of organic solvents, 1,4-dioxane–water
binary mixtures and in the microheterogeneous media of
sodium dodecyl sulphate (SDS), cetyl trimethyl ammonium
bromide (CTAB) and Triton-X100 micelles. Moreover, to
examine the usefulness of these benzonaphthyridines as a
fluorescence probes for proteins, it has been covalently
attached to human serum albumin (HSA) and bovine serum
albumin (BSA).
Synthesis
Synthesis of pyrimido[4,5-b]quinoline (3a–d)
Method1. A solution of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001mol)
and formamide (2a; 0.001mol) in ethanolic potassium hydroxide solution
(10 mL, 2%) was refluxed for 3 h. Completion of the reaction was monitored
by thin-layer chromatography (TLC). The mixture was then cooled to room
temperature; the separated solid product was collected by suction filtration,
washed with pet-ether, dried and recrystallized from ethanol.
Method 2. The compound 1a-b (0.001 mmol) and the corresponding
formamide (2a; 0.001 mol) was heated at 175–180 ꢀC for 1H (TLC check).
On cooling, the molten mass was stirred in ethanol (2 mL) for 15 min. The
solid obtained on cooling was collected by filtration, washed with cold
ethanol and crystallized from ethanol.
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Interaction of benzo[b][1,8]naphthyridines with HSA and BSA
Method 3. A solution of 1a-b (0.001 mol) and the corresponding formam-
ide (2a; 0.001 mol) was heated in diphenyl ether at about 190–210 ꢀC for
2.5 h (monitored by TLC check). On cooling, the molten mass was stirred
in ethanol (2 mL) for 15 min. The solid obtained on cooling was collected
by filtration, washed with cold ethanol and recrystallized from ethanol.
the separated solid product was collected by suction filtration, washed
with pet-ether, dried and recrystallized from ethanol.
3-Phenylbenzo[b][1,8]naphthyridin-2-amine (5a). Yield: 0.222 g (81%),
recrystallized from ethanol to afford faint yellow prisms; m.p.
218–221 ꢀC. IR (KBr): 3321 m, 3259 m, 3014 m, 2977 m, 2968 w, 1624 s cm–1.
1H-NMR (300 MHz CDCl3) d: 6.68 (bs, 2H, NH2), 7.21–7.33 (m, 5H, Ar-H), 7.49
(dd, 1H, J=8.0 and 8.4 Hz, Ar-H), 7.61 (dd, 1H, J=8.4 and 8.5 Hz, Ar-H), 8.13
(d, 1H, J=8.0 Hz, Ar-H), 8.29 (d, 1H, J=8.5 Hz, Ar-H), 8.63 (s, 1H, Ar-H), 8.94
(s, 1H, Ar-H). Anal. calcd. for C18H13N3 (271.32): C, 79.70; H, 4.79; N, 15.49%;
found, C, 79.74; H, 4.80; N, 15.45%.
Method 4. A solution of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001 mol)
and formamide (2a; 0.001mol) in aqueous HCl solution (10mL, 2%) was
refluxed for 6h. Completion of the reaction was monitored by TLC. The
mixture was then cooled at room temperature. The separated solid product
was collected by suction filtration, washed with n-hexane, dried and recrys-
tallized from ethanol.
7-Methoxy-3-phenylbenzo[b][1,8]naphthyridin-2-amine (5b). Yield: 0.254 g
(84%), recrystallized from ethanol to afford yellow needles; m.p.
227–229 ꢀC. IR (KBr): 3349 m, 3278 m, 3029 m, 2951 m, 2904 m, 1617 m,
Method 5. A solution of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001 mol)
and corresponding amide (2a; 0.001mol) in the catalytic amount of
SnCl.22H2O reaction mass was refluxed for 15–140 min. The reaction was
monitored with the help of TLC. After completion of the reaction (TLC
check), the reaction mass was poured over crushed ice. The reaction mass
was stirred and neutralized with aq. NaHCO3 solution, extracted with ethyl
acetate (25mL) and concentrated over a rotary evaporator. The solid was
collected by suction filtration and recrystallized from cold ethanol.
1022 w cm–1 1H-NMR (300 MHz CDCl3) d: 4.09 (s, 3H, OCH3), 6.89 (bs,
.
2H, NH2), 7.08–7.21 (m, 5H, Ar-H), 7.40 (d, 1H, J = 8.8 Hz, Ar-H), 7.69
(s, 1H, Ar-H), 8.14 (d, 1H, J = 8.8 Hz, Ar-H), 8.51 (s, 1H, Ar-H), 8.84 (s, 1H,
Ar-H). 13 C-NMR (75 MHz CDCl3) d: 59.2, 112.4, 120.3, 122.9, 124.1, 126.9
(2 Â Cs), 128.5, 129.6 (2 Â Cs), 130.1, 131.7, 137.2, 137.9, 138.4, 143.6,
156.5, 157.8, 158.3. Anal. calcd. for C19H15N3O (301.34): C, 75.74; H, 4.98;
N, 13.95%; found, C, 75.76; H, 4.98; N, 13.99%.
Method 6. A mixture of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001 mol)
and formamide (2a; 0.001mol) and conc. HCl (2–3 drops, AR grade) was
taken in a borosil round-bottomed flask attached to a condenser and irradi-
ated in an unmodified domestic microwave oven at 70W for 1.5–12min
(monitored by TLC). The resultant mixture was cooled to room temperature;
cold water was added, the mixture was neutralized to pH 7 with aq.
NaHCO3 solution (10% v/v). The separated solid was filtered by suction,
washed with cold water, dried and recrystallized using ethanol.
Synthesis of 2,3-dihydro-1H-benzo[b]cyclopenta[g][1,8]naphthyridine (7a-d).
The mixture of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001 mol) and
cyclopentanone 6a (0.001 mol) in ethanolic potassium hydroxide solution
(10 mL, 2%) was refluxed for 5 h. Completion of the reaction was moni-
tored by TLC. The mixture was then cooled to room temperature; the
separated solid product was collected by suction filtration, washed with
pet-ether, dried and recrystallized from ethanol.
Pyrimido[4,5-b]quinoline (3a). Yield: 0.155 g (85%), recrystallized from eth-
anol to afford a faint yellow prisms; m.p. 164–167 ꢀC. IR (KBr): 3012 m,
2,3-Dihydro-1H-benzo[b]cyclopenta[g][1,8]naphthyridine (7a). Yield: 0.178 g
2968 m, 1618 s cm–1 1H-NMR (300 MHz CDCl3) d: 7.34 (dd, 1H, J = 8.2
.
(80%), recrystallized from ethanol to afford a faint yellow solid; m.p.
171–173 ꢀC. IR (KBr): 3012 m, 2968 m, 2912 w, 1610 s cm–1 1H-NMR
.
and 8.7 Hz, Ar-H), 7.79 (dd, 1H, J = 8.7 and 8.1 Hz, Ar-H), 8.09 (d, 1H,
J = 8.2 Hz, Ar-H), 8.36 (d, 1H, J = 8.1 Hz, Ar-H), 8.69 (s, 1H, Ar-H), 8.78 (s,
1H, Ar-H), 9.19 (s, 1H, Ar-H). Anal. calcd. for C11H7N3 (181.19): C, 72.92;
H, 3.86; N, 23.20%; found, C, 72.94; H, 3.83; N, 23.19%.
(300 MHz CDCl3) d: 1.95 (m, 2H, CH2), 2.49 (t, 2H, J=7.4 Hz, CH2), 3.09 (t, 2H,
J= 6.8 Hz, CH2), 7.34 (dd, 1H, J=7.9 and 8.3 Hz, Ar-H), 7.74 (dd, 1H, J=8.3
and 8.4 Hz, Ar-H), 8.09 (d, 1H, J=7.9Hz, Ar-H), 8.36 (d, 1H, J=8.4 Hz, Ar-H),
8.78 (s, 1H, Ar-H), 9.19 (s, 1H, Ar-H). Anal. calcd. for C15H12N2 (220.27): C,
81.81; H, 5.45; N, 12.72%; found, C, 81.80; H, 5.48; N, 12.70%.
7-Methoxypyrimido[4,5-b]quinoline (3b). Yield: 0.172 g (81%), recrystal-
lized from ethanol to afford faint yellow needles; m.p. 175–178 ꢀC. IR
(KBr): 3023 m, 2960 m, 1620 s, 1031 s cm–1 1H-NMR (300 MHz CDCl3) d:
.
8-Methoxy-2,3-dihydro-1H-benzo[b]cyclopenta[g][1,8]naphthyridine (7b).
Yield: 0.211 g (84%), recrystallized from ethanol to afford faint yellow
needles; m.p. 184–186 ꢀC. IR (KBr): 3019 m, 2972 m, 2929m, 1619s,
1026 s cm–1.1H-NMR (300 MHz CDCl3) d: 1.88 (m, 2H, CH2), 2.37 (t, 2H,
J= 7.1 Hz, CH2), 3.14 (t, 2H, J = 6.9Hz, CH2), 4.02 (s, 3H, OCH3), 7.43 (d, 1H,
J= 7.7Hz, Ar-H), 7.51 (s, 1H, Ar-H), 8.24 (d, 1H, J = 7.7Hz, Ar-H), 9.21 (s, 1H,
Ar-H), 9.44 (s, 1H, Ar-H). MS (70eV) m/z (%): 250 [M+] (83), 206 (37), 125
(19), 77 (22), 44 (85), 32 (47). Anal. calcd. for C16H14N2O (250.29): C, 76.80;
H, 5.60; N, 11.20%; found, C, 76.82; H, 5.62; N, 11.22%.
4.02 (s, 3H, OCH3), 7.43 (d, 1H, J = 9.0 Hz, Ar-H), 7.51 (s, 1H, Ar-H), 8.24
(d, 1H, J = 9.0 Hz, Ar-H), 8.68 (s, 1H, Ar-H), 9.21 (s, 1H, Ar-H), 9.44 (s, 1H,
Ar-H). 13C-NMR (75 MHz CDCl3) d: 59.2, 112.7, 125.4, 127.5, 130.6, 131.7,
136.7, 143.7, 148.7, 157.1, 158.9, 160.1. Anal. calcd. for C12H9N3O
(211.22): C, 68.24; H, 4.26; N, 19.90%; found, C, 68.25; H, 4.23; N, 19.93%.
2-Phenylpyrimido[4,5-b]quinoline (3c). Yield: 0.217 g (84%), recrystallized
from ethanol to afford a yellow solid; m.p. 194–196 ꢀC. IR (KBr): 3010 m,
2958 m, 1628 s cm–1 1H-NMR (300 MHz CDCl3) d: 7.12–7.40 (m, 5H,
.
1,2,3,4-Tetrahydrodibenzo[b,g][1,8]naphthyridine (7c). Yield: 0.202 g (86%),
recrystallized from ethanol to afford a yellow solid; m.p. 206–209 ꢀC. IR
Ar-H), 7.48 (dd, 1H, J = 8.3 and 8.5 Hz, Ar-H), 7.84 (dd, 1H, J = 8.5 and
7.9 Hz, Ar-H), 8.12 (d, 1H, J = 8.3 Hz, Ar-H), 8.29 (d, 1H, J = 7.9 Hz, Ar-H),
8.58 (s, 1H, Ar-H), 9.06 (s, 1H, Ar-H). Anal. calcd. for C17H11N3 (257.29): C,
79.37; H, 4.28; N, 16.34%; found, C, 79.34; H, 4.32; N, 16.35%.
(KBr): 2994 m, 2971 m, 2917 m, 1622 s cm–1 1H-NMR (300MHz CDCl3)
.
d: 1.67–1.74 (m, 4H, CH2), 2.60 (t, 2H, J= 6.8Hz, CH2), 2.94 (t, 2H,
J= 6.9 Hz, CH2), 7.27 (dd, 1H, J= 8.2 and 8.6Hz, Ar-H), 7.61 (dd, 1H, J = 8.6
and 8.1Hz, Ar-H), 7.97 (d, 1H, J = 8.2Hz, Ar-H), 8.31 (d, 1H, J= 8.1 Hz,
Ar-H), 8.68 (s, 1H, Ar-H), 9.17 (s, 1H, Ar-H). Anal. calcd. for C16H14N2
(234.30): C, 82.05; H, 5.98; N, 11.96%; found, C, 82.03; H, 6.00; N, 11.98%.
7-Methoxy-2-phenylpyrimido[4,5-b]quinoline (3d). Yield: 0.236 g (82%),
recrystallized from ethanol to afford a yellow solid; m.p. 201–204 ꢀC. IR
(KBr): 3007 m, 2960 m, 1616 m, 1099 s cm–1 1H-NMR (300 MHz CDCl3) d:
.
3.98 (s, 3H, OCH3), 7.12–7.29 (m, 5H, Ar-H), 7.39 (d, 1H, J = 9.2 Hz, Ar-H),
7.56 (s, 1H, Ar-H), 8.20 (d, 1H, J = 9.2 Hz, Ar-H), 8.51 (s, 1H, Ar-H), 9.04 (s,
1H, Ar-H). 03C-NMR (75 MHz CDCl3) d: 58.9, 111.9, 122.5, 124.7, 127.5 (2
 Cs), 128.2, 129.4 (2  Cs), 129.9, 131.9, 133.1, 136.7, 144.9, 151.2,
157.7, 158.2, 163.1. Anal. calcd. for C18H13N3O (287.32): C, 75.26; H, 4.52;
N, 14.63%; found, C, 75.25; H, 4.50; N, 14.65%.
9-Methoxy-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine (7d). Yield:
0.231 g (87%), recrystallized from ethanol to afford yellow needles;
m.p. 231–234 ꢀC. IR (KBr): 3006 m, 2968 m, 1618 s, 1011 s cm–1.1H-NMR
(300 MHz CDCl3) d: 1.55–1.68 (m, 4H, CH2), 2.74 (t, 2H, J = 7.1 Hz, CH2),
3.01 (t, 2H, J = 6.5 Hz, CH2), 4.02 (s, 3H, OCH3), 7.38 (d, 1H, J = 8.8 Hz,
Ar-H), 7.58 (s, 1H, Ar-H), 8.17 (d, 1H, J = 8.8 Hz, Ar-H), 9.02 (s, 1H, Ar-H),
9.31 (s, 1H, Ar-H). 13C-NMR (75 MHz CDCl3) d: 21.9, 22.2, 32.5, 34.7,
58.1, 112.9, 123.4, 125.1, 129.1, 131.2, 133.5, 135.1, 137.8, 144.7, 156.2,
157.9, 159.9. MS (70 eV) m/z (%): 264 [M+] (72), 220 (42), 132 (29), 110
(33), 44 (77), 32 (68). Anal. calcd. for C17H16N2O (264.32): C, 77.27; H,
6.06; N, 10.60%; found, C, 77.29; H, 6.09; N, 10.58%.
Synthesis of 3-Phenylbenzo[b][1,8]naphthyridin-2-amine (5a-b). The mix-
ture of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001 mol) and benzyl-
cyanide (4; 0.001 mol) in ethanolic potassium hydroxide solution
(10 mL, 2%) was refluxed for 6 h. Completion of the reaction was
monitored by TLC. The mixture was then cooled to room temperature;
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D. P. Shelar et al.
Synthesis of 4-methyl-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine (9a-b).
A mixture of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001mol) and
2-methylcyclohexanone (8) (0.001 mol) in ethanolic potassium hydroxide
solution (10 mL, 2%) was refluxed for 6 h. Completion of the reaction was
monitored by TLC. The mixture was then cooled to room temperature; the
separated solid product was collected by suction filtration, washed with cold
methanol, dried and recrystallized from ethanol.
9-Methoxy-3,3-dimethyl-3,4-dihydrodibenzo[b,g][1,8]naphthyridin-1(2H)-
one (11d). Yield: 0.256 g (83%), recrystallized from ethanol to afford
green needles; m.p. 229–231 ꢀC. IR (KBr): 3018 m, 2966 m, 2913 m,
1703 s, 1604 s, 1010 s cm–1.1H-NMR (300 MHz CDCl3) d: 1.25 (s, 6H,
CH3), 2.39 (s, 2H, CH2), 2.94 (s, 2H, CH2), 4.09 (s, 3H, OCH3), 7.36 (d,
1H, J = 8.8 Hz, Ar-H), 7.51 (s, 1H, Ar-H), 8.24 (d, 1H, J = 8.8 Hz, Ar-H),
9.28 (s, 1H, Ar-H), 9.46 (s, 1H, Ar-H). 13 C-NMR (75 MHz CDCl3) d: 23.4,
25.7, 30.6, 44.8, 50.7, 58.7, 113.4, 123.8, 125.3, 131.2, 132.7, 134.1,
136.9, 138.3, 144.1, 156.6, 158.7, 166.2, 197.7. MS (70 eV) m/z (%): 307
[M + H]+ (100), 278(29), 250(22), 179(18), 55(12), 41(26). Anal. calcd.
for C19H18N2O2 (306.36): C, 74.50; H, 5.88; N, 9.15%; found, C, 74.52;
H, 5.90; N, 9.14%.
4-Methyl-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine (9a). Yield:
0.204 g (82%), recrystallized from ethanol to afford yellow prisms;
m.p. 211–213 ꢀC. IR (KBr): 3015 m, 2968 m, 2912 m, 1618 s cm–1
.
1H-NMR (300 MHz CDCl3) d: 1.52 (d, 3H, J = 5.5 Hz, CH3), 1.89 (m, 2H,
CH2), 2.04 (m, 2H, CH2), 2.49 (t, 2H, J = 7.4 Hz, CH2), 3.26 (m, 1H, CH),
7.16 (dd, 1H, J = 7.9 and 8.3 Hz, Ar-H), 7.77 (dd, 1H, J = 8.3 and 8.5 Hz,
Ar-H), 8.14 (d, 1H, J = 7.9 Hz, Ar-H), 8.27 (d, 1H, J = 8.5 Hz, Ar-H), 8.82
(s, 1H, Ar-H), 9.07 (s, 1H, Ar-H). 13C-NMR (75 MHz CDCl3) d: 19.1, 20.5,
28.7, 29.9, 31.2, 119.9, 126.1, 127.8, 128.5, 129.5, 131.1, 133.9, 135.3,
138.2, 149.5, 158.1, 161.5. Anal. calcd. for C17H16N2 (248.32): C,
82.25; H, 6.45; N, 11.29%; found, C, 82.27; H, 6.46; N, 11.30%.
Synthesis of 7-(3,4-dichlorophenyl)-7,12-dihydrobenzo[b]naphtho[2,3-g][1,8]
naphthyridine (13a-b). The mixture of 2-aminoquinoline-3-carbaldehyde
(1a-b; 0.001 mol) and cetraline (12; 0.001mol) in ethanolic potassium
hydroxide solution (10 mL, 2%) was refluxed for 3 h. Completion of the
reaction was monitored by TLC. The mixture was then cooled to room
temperature; the separated solid product was collected by suction filtra-
tion, washed with pet-ether, dried and recrystallized from ethanol.
9-Methoxy-4-methyl-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine (9b).
5-(3,4-Dichlorophenyl)-5,6-dihydrobenzo[b]naphtho[2,1-g][1,8]naphthyri-
dine (13a). Yield: 0.358 g (84%), recrystallized from ethanol to afford a yel-
low solid; m.p. 284–286 ꢀC. IR (KBr): 2991 m, 2959 m, 1620 s cm–1. 1H-NMR
(300 MHz DMSO-d6) d: 3.84 (d, 2H, J =5.8 Hz, CH2), 5.09 (t, 1H, J=5.8 Hz, CH2),
6.89–7.09 (m, 4H, Ar-H), 7.15 (dd, 1H, J=8.6 and 3.2 Hz, Ar-H), 7.24 (d, 1H,
J=3.2 Hz, Ar-H), 7.39 (dd, 1H, J=8.0 and 8.5 Hz, Ar-H), 7.49 (d, 1H, J=8.6 Hz,
Ar-H), 7.70 (dd, 1H, J=8.5 and 8.1 Hz, Ar-H), 8.14 (d, 1H, J=8.0 Hz, Ar-H), 8.44
(d, 1H, J=8.1 Hz, Ar-H), 8.81 (s, 1H, Ar-H), 9.08 (s, 1H, Ar-H). 13C-NMR (75 MHz
DMSO-d6) d: 38.3, 43.5, 120.2, 121.9, 122.5, 123.1, 124.9, 125.1, 125.8, 127.2,
128.1, 128.7, 129.2, 129.8, 131.4, 132.9, 133.9, 135.6, 136.4, 137.1, 137.9, 141.4,
144.9, 148.3, 159.3, 162.7. Anal. calcd. for C26H16Cl2N2 (427.33): C, 73.23; H,
3.75; N, 6.57%; found, C, 73.20; H, 3.74; N, 6.55%.
Yield: 0.239 g (85%), recrystallized from ethanol to afford a yellow solid; m.p.
1
171–173 ꢀC. IR (KBr): 3021 m, 2959 m, 2926 m, 1622 s, 1029 s cm–1. H-NMR
(300 MHz CDCl3) d: 1.47 (d, 3H, J=5.8 Hz, CH3), 1.84 (m, 2H, CH2), 2.12
(m, 2H, CH2), 2.45 (t, 2H, J=6.7 Hz, CH2), 3.12 (m, 1H, CH), 3.98 (s, 3H, OCH3),
7.33 (d, 1H, J=8.3 Hz, Ar-H), 7.44 (s, 1H, Ar-H), 7.97 (d, 1H, J=8.3 Hz, Ar-H),
9.32 (s, 1H, Ar-H), 9.49 (s, 1H, Ar-H). MS (70eV) m/z (%): 278 [M+] (100), 263
(92), 249 (47), 206 (22), 131 (12), 109 (22). Anal. calcd. for C18H18N2O
(278.35): C, 77.69; H, 6.47; N, 10.07%; found, C, 77.70; H, 6.48; N, 10.09%.
Synthesis of 3,4-dihydrodibenzo[b,g][1,8]naphthyridin-1(2H)-one (11a-d). A
mixture of 1a-b (0.001mol) and dimedone (10a-b; 0.001mol) was heated
at 180–185 ꢀC for 15 min. On cooling, the molten mass was stirred in
methanol (10mL) for 15min. The separated solid was collected by suction
filtration and recrystallized from ethanol.
5-(3,4-Dichlorophenyl)-10-methoxy-5,6-dihydrobenzo[b]naphtho[2,1-g][1,8]
naphthyridine (13b). Yield: 0.391 g (85%), recrystallized from ethanol to af-
ford a yellow solid; m.p. 291–293 ꢀC. IR (KBr): 3011 m, 2975 m, 2934 m,
1609 s, 1029 s cm–1 1H-NMR (300 MHz DMSO-d6) d: 3.77 (d, 2H,
.
3,4-Dihydrodibenzo[b,g][1,8]naphthyridin-1(2H)-one (11a). Yield: 0.199 g
(80%), recrystallized from ethanol to afford a faint yellow solid; m.p.
239–241 ꢀC. IR (KBr): 3071 m, 2977 m, 2931 w, 1705 s, 1604 s cm–1. 1H-NMR
(300MHz CDCl3) d: 1.88 (m, 2H, CH2), 2.56 (t, 2H, J= 6.5 Hz, CH2), 2.94 (t,
2H, J= 6.7 Hz, CH2), 7.21 (dd, 1H, J= 8.2 and 8.6 Hz, Ar-H), 7.55 (dd, 1H,
J =8.6 and 8.1 Hz, Ar-H), 8.11 (d, 1H, J= 8.2Hz, Ar-H), 8.47 (d, 1H, J= 8.1 Hz,
Ar-H), 8.68 (s, 1H, Ar-H), 9.19 (s, 1H, Ar-H). Anal. calcd. for C16H12N2O
(248.28): C, 77.41; H, 4.83; N, 11.29%; found, C, 77.40; H, 4.80; N, 11.31%.
J = 5.9 Hz, CH2), 4.04 (s, 3H, OCH3), 4.88 (t, 1H, J = 5.9 Hz, CH2), 6.94–7.13
(m, 4H, Ar-H), 7.19 (dd, 1H, J = 8.5 and 2.9 Hz, Ar-H), 7.29 (d, 1H,
J = 2.9 Hz, Ar-H), 7.37 (d, 1H, J = 8.5 Hz, Ar-H), 7.47 (d, 1H, J = 8.9 Hz, Ar-H),
7.62 (s, 1H, Ar-H), 8.24 (d, 1H, J = 8.9 Hz, Ar-H), 9.21 (s, 1H, Ar-H), 9.31 (s,
1H, Ar-H). MS (70 eV) m/z (%): 456 [M+] (86), 458 [M + 2] (59), 460 [M + 4]
(9), 441 (29), 268 (31), 171 (47), 193 (40), 44 (84), 32 (49). Anal. calcd. for
C27H18Cl2N2O (457.35): C, 71.05; H, 3.94; N, 6.14%; found, C, 71.07; H, 3.91; N, 6.16%.
9-Methoxy-3,4-dihydrodibenzo[b,g][1,8]naphthyridin-1(2H)-one (11b).
Yield: 0.227 g (81%), recrystallized from ethanol to afford yellow nee-
dles; m.p. 184–186 ꢀC. IR (KBr): 3009 m, 2970 m, 2918 m, 1698 s, 1615 s,
1019 s cm–1.1H-NMR (300 MHz CDCl3) d: 1.94 (m, 2H, CH2), 2.47 (t, 2H,
J = 7.2 Hz, CH2), 2.83 (t, 2H, J = 6.6 Hz, CH2), 4.05 (s, 3H, OCH3), 7.37 (d,
1H, J = 9.0 Hz, Ar-H), 7.55 (s, 1H, Ar-H), 8.20 (d, 1H, J = 9.0 Hz, Ar-H), 9.27
(s, 1H, Ar-H), 9.38 (s, 1H, Ar-H). 13C-NMR (75 MHz CDCl3) d: 21.8, 31.7,
39.7, 58.3, 113.2, 123.3, 124.9, 128.9, 130.5, 134.5, 136.5, 137.2,
144.7, 156.7, 158.1, 166.5, 197.2. MS (70 eV) m/z (%): 279 [M + H]+
(100), 236 (22), 202 (48), 174 (37), 131 (38), 77 (57), 51(49). Anal. calcd.
for C17H14N2O2 (278.30): C, 73.38; H, 5.03; N, 10.07%; found, C, 73.40;
H, 5.05; N, 10.08%.
Synthesis of 3-methoxy-5,6-dihydrobenzo[b]naphtho[2,1-g] [1,8]naphthyri-
dine (15a–b). The mixture of 2-aminoquinoline-3-carbaldehyde (1a–b;
0.001 mol) and 6-methoxy-tetralone (14; 0.001 mol) in ethanolic potas-
sium hydroxide solution (10 mL, 2%) was refluxed for 2.5 h. Completion
of the reaction was monitored by TLC. The mixture was then cooled to
room temperature; the separated solid product was collected by suction
filtration, washed with n-hexane, dried and recrystallized from ethanol.
3-Methoxy-5,6-dihydrobenzo[b]naphtho[2,1-g][1,8]naphthyridine (15a).
Yield: 0.260 g (83%), recrystallized from ethanol to afford a faint yellow
solid; m.p. 275–277 ꢀC. IR (KBr): 2986 m, 2969 m, 2911 m, 1624 s, 1029 s
cm–1 1H-NMR (300 MHz CDCl3) d: 2.26 (d, 2H, J = 4.8 Hz, CH2), 2.39 (d,
.
2H, J = 4.8 Hz, CH2), 3.94 (s, 3H, OCH3), 6.89 (dd, 1H, J = 7.7 and 3.4 Hz,
Ar-H), 7.08 (d, 1H, J = 3.4 Hz, Ar-H), 7.16 (d, 1H, J = 7.7 Hz, Ar-H), 7.34
(dd, 1H, J = 7.9 and 8.7 Hz, Ar-H), 7.73 (dd, 1H, J = 8.7 and 8.0 Hz, Ar-H),
8.18 (d, 1H, J = 7.9 Hz, Ar-H), 8.31 (d, 1H, J = 8.0 Hz, Ar-H), 8.85 (s, 1H,
Ar-H), 9.14 (s, 1H, Ar-H). Anal. calcd. for C21H16N2O (312.37): C, 80.76;
H, 5.12; N, 8.97%; found, C, 80.75; H, 5.12; N, 8.99%.
3,3-Dimethyl-3,4-dihydrodibenzo[b,g][1,8]naphthyridin-1(2H)-one (11c).
Yield: 0.229 g (82%), recrystallized from ethanol to afford a faint green
solid; m.p. 201–203 ꢀC. IR (KBr): 3033 m, 2960 m, 2914 m, 1696 s, 1626 s
cm–1. 1H-NMR (300 MHz CDCl3) d: 1.19 (s, 6H, CH3), 2.44 (s, 2H, CH2), 2.89
(s, 2H, CH2), 7.39 (dd, 1H, J = 7.9 and 8.5 Hz, Ar-H), 7.70 (dd, 1H, J = 8.5
and 8.1 Hz, Ar-H), 8.09 (d, 1H, J = 7.9 Hz, Ar-H), 8.30 (d, 1H, J = 8.1 Hz,
Ar-H), 8.71 (s, 1H, Ar-H), 9.10 (s, 1H, Ar-H). MS (70 eV) m/z (%): 277
[M + H]+ (100), 248 (22), 201 (28), 183 (27), 152 (22), 77 (52), 51 (41),
31 (63). Anal. calcd. for C18H16N2O (276.33): C, 78.26; H, 5.79; N,
10.14%; found, C, 78.29; H, 5.80; N, 10.15%.
3,10-Dimethoxy-5,6-dihydrobenzo[b]naphtho[2,1-g][1,8]naphthyridine (15b).
Yield: 0.285 g (83%), recrystallized from ethanol to afford faint green solid;
m.p. 297–299 ꢀC. IR (KBr): 3017 m, 2979 m, 2928 m, 1609 s, 1043 s cm–1
.
1H-NMR (300 MHz DMSO-d6) d: 2.35 (d, 2H, J = 5.3Hz, CH2), 2.51 (d, 2H,
wileyonlinelibrary.com/journal/luminescence
Copyright © 2011 John Wiley & Sons, Ltd. Luminescence 2011

Interaction of benzo[b][1,8]naphthyridines with HSA and BSA
J= 5.3Hz, CH2), 3.90 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 6.72 (dd, 1H, J = 8.2
and 3.1 Hz, Ar-H), 7.06 (d, 1H, J = 3.1 Hz, Ar-H), 7.18 (d, 1H, J = 8.2 Hz,
Ar-H), 7.33 (d, 1H, J= 8.5Hz, Ar-H), 7.42 (s, 1H, Ar-H), 8.31 (d, 1H,
J= 8.5Hz, Ar-H), 8.68 (s, 1H, Ar-H), 9.21 (s, 1H, Ar-H). 13C-NMR (75 MHz
DMSO-d6) d: 29.1, 34.7, 58.8, 59.7, 114.3, 117.2, 118.1, 124.5, 125.3, 126.1,
128.3, 129.8, 131.7, 133.8, 135.2, 137.1, 138.2, 144.1, 156.2, 158.3, 159.1,
162.5. Anal. calcd. for C22H18N2O2 (342.39): C, 77.19; H, 5.26; N, 8.18%;
found, C, 77.20; H, 5.25; N, 8.21%.
Synthesis of 2-methyl-1,2,3,4-tetrahydroquinolino[2,3-b][1,6]naphthyridine
(21a-f). The mixture of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001mol)
and piperidone (20a-c; 0.001 mol) in ethanolic potassium hydroxide solution
(10 mL, 2%) was refluxed for 3 h. Completion of the reaction was monitored
by TLC. The mixture was then cooled to room temperature; the separated
solid product was collected by suction filtration, washed with pet-ether,
dried and recrystallized from ethanol.
2-Methyl-1,2,3,4-tetrahydroquinolino[2,3-b][1,6]naphthyridine (21a). Yield:
0.207 g (83%), recrystallized from ethanol to afford a yellow solid; m.p.
Synthesis of 5′,5′-Dimethyl-3,4-dihydro-1H-spiro[dibenzo[b,g][1,8]naphthyridine-
2,2′-[1,3]dioxane] (17a-b). The mixture of 2-aminoquinoline-3-carbaldehyde
(1a-b; 0.001 mol) and 3,3-dimethyl-1,5-dioxaspiro[5,5]undecan-9-one (16;
0.001 mol) in ethanolic potassium hydroxide solution (10 mL, 2%) was
refluxed for 6 h. Completion of the reaction was monitored by TLC. The mix-
ture was then cooled to room temperature; the separated solid product was
collected by suction filtration, washed with pet-ether, dried and recrystallized
from ethanol.
256–258 ꢀC. IR (KBr): 3007 m, 2974 m, 2942 m, 1616 s cm–1 1H-NMR
.
(300 MHz CDCl3) d: 2.42 (s, 3H, CH3), 2.95 (t, 2H, J = 6.5 Hz, CH2), 3.12 (t,
2H, J = 6.5 Hz, CH2), 3.89 (s, 2H, CH2), 7.38 (dd, 1H, J = 8.1 and 8.5 Hz,
Ar-H), 7.67 (dd, 1H, J = 8.5 and 8.6 Hz, Ar-H), 8.16 (d, 1H, J = 8 .1 Hz,
Ar-H), 8.27 (d, 1H, J = 8.6 Hz, Ar-H), 8.67 (s, 1H, Ar-H), 9.12 (s, 1H, Ar-H).
Anal. calcd. for C16H15N3 (249.31): C, 77.10; H, 6.02; N, 16.86%; found, C,
77.12; H, 6.04; N, 16.88%.
9-Methoxy-2-methyl-1,2,3,4-tetrahydroquinolino[2,3-b][1,6]naphthyridine
(21b). Yield: 0.236 g (84%), recrystallized from ethanol to afford a faint brown
solid; m.p. 267–269 ꢀC. IR (KBr): 2994 m, 2945 m, 2911 m, 1618 s, 1029 s cm–1.
1H-NMR (300 MHz CDCl3) d: 2.38 (s, 3H, CH3), 2.70 (t, 2H, J=5.8 Hz, CH2), 3.14
(t, 2H, J=5.8 Hz, CH2), 4.03 (s, 3H, OCH3), 4.17 (s, 2H, CH2), 7.37 (d, 1H,
J=8.8 Hz, Ar-H), 7.56 (s, 1H, Ar-H), 8.17 (d, 1H, J=8.8 Hz, Ar-H), 8.77 (s, 1H,
Ar-H), 9.17 (s, 1H, Ar-H). MS (70eV) m/z (%): 278 [M-H]+ (100), 236 (33), 202
(37), 174 (28), 131 (42), 117 (22), 77 (12), 44 (19). Anal. calcd. for C17H17N3O
(279.34): C, 73.11; H, 6.09; N, 15.05%; found, C, 73.12; H, 6.11; N, 15.07%.
5′,5′-Dimethyl-3,4-dihydro-1H-spiro[dibenzo[b,g][1,8]naphthyridine-2,2′-[1,3]
dioxane] (17a). Yield: 0.271 g (81%), recrystallized from ethanol to afford
yellow needles; m.p. 231–233 ꢀC. IR (KBr): 3022 m, 2974 m, 1613s, 1033 s
cm–1 1H-NMR (300 MHz CDCl3) d: 1.21 (s, 6H, (CH3)2), 2.35 (t, 2H,
.
J = 4.5 Hz, CH2), 2.67 (s, 2H, CH2), 3.02 (t, 2H, J = 4.5 Hz, CH2), 4.31 (s, 4H,
CH2), 7.44 (dd, 1H, J = 8.3 and 8.8 Hz, Ar-H), 7.81 (dd, 1H, J = 8.8 and
8.0 Hz, Ar-H), 8.12 (d, 1H, J = 8.3Hz, Ar-H), 8.30 (d, 1H, J = 8.0 Hz, Ar-H),
8.88 (s, 1H, Ar-H), 9.14 (s, 1H, Ar-H). Anal. calcd. for C21H22N2O2 (334.41):
C, 75.44; H, 6.58; N, 8.38%; found, C, 75.49; H, 6.59; N, 8.42%.
1-(3,4-Dihydroquinolino[2,3-b][1,6]naphthyridin-2(1H)-yl)ethanone (21c).
Yield: 0.237 g (85%), recrystallized from ethanol to afford a faint brown
solid; m.p. 214–217 ꢀC. IR (KBr): 3019 m, 2966 w, 2928 m, 1679 s, 1610 s cm–1.
1H-NMR (300 MHz CDCl3) d: 2.13 (s, 3H, CH3), 3.22 (t, 2H, J=6.6 Hz, CH2), 3.49
(t, 2H, J=6.6 Hz, CH2), 4.73 (s, 2H, CH2), 7.42 (dd, 1H, J=8.4 and 8.5 Hz, Ar-H),
7.68 (dd, 1H, J=8.5 and 8.3 Hz, Ar-H), 8.11 (d, 1H, J=8.4 Hz, Ar-H), 8.39 (d,
1H, J=8.3 Hz, Ar-H), 8.82 (s, 1H, Ar-H), 9.08 (s, 1H, Ar-H). 13C-NMR (75 MHz
CDCl3) d: 23.5, 35.2, 41.3, 44.2, 121.7, 126.2, 126.9, 127.6, 129.1, 129.9, 130.5,
133.7, 138.1, 148.1, 154.1, 157.2, 173.3. Anal. calcd. for C17H15N3O (277.32): C,
73.64; H, 5.41; N, 15.16%; found, C, 73.61; H, 5.40; N, 15.16%.
9-Methoxy-5′,5′-dimethyl-3,4-dihydro-1H-spiro[dibenzo[b,g][1,8]naphthyridine-
2,2′-[1,3]dioxane] (17b). Yield: 0.298 g (81%), recrystallized from ethanol to
afford a yellow solid; m.p. 214–216ꢀC. IR (KBr): 3017 m, 2970 m, 1615 s,
1041 s cm–1 1H-NMR (300 MHz CDCl3) d: 1.17 (s, 6H, (CH3)2), 2.42 (t, 2H,
.
J= 5.3Hz, CH2), 2.71 (s, 2H, CH2), 2.94 (t, 2H, J= 5.3Hz, CH2), 4.07 (s, 3H,
OCH3), 4.38 (s, 4H, CH2), 7.29 (d, 1H, J= 8.7Hz, Ar-H), 7.49 (s, 1H, Ar-H),
8.26 (d, 1H, J = 8.7Hz, Ar-H), 8.73 (s, 1H, Ar-H), 9.17 (s, 1H, Ar-H). 13C-NMR
(75 MHz CDCl3) d: 18.4, 19.2, 21.3, 28.4, 30.5, 41.5, 58.9, 71.7 (2 Â Cs), 88.8,
114.1, 123.1, 125.7, 129.8, 131.7, 133.4, 136.0, 138.3, 145.3, 158.2, 159.5,
161.5. Anal. calcd. for C22H24N2O3 (364.44): C, 72.52; H, 6.59; N, 7.69%; found,
C, 72.55; H, 6.60; N, 7.67%.
1-(9-Methoxy-3,4-dihydroquinolino[2,3-b][1,6]naphthyridin-2(1H)-yl)etha-
none (21d). Yield: 0.269 g (87%), recrystallized from ethanol to afford a
yellow solid; m.p. 244–247 ꢀC. IR (KBr): 3012 m, 2959 m, 2936 w, 1683 s,
1607 s, 1024 s cm–1. 1H-NMR (300 MHz CDCl3) d: 2.09 (s, 3H, CH3), 2.78 (t,
2H, J = 6.4 Hz, CH2), 3.19 (t, 2H, J = 6.4 Hz, CH2), 4.05 (s, 3H, OCH3), 4.81 (s,
2H, CH2), 7.36 (d, 1H, J = 7.9 Hz, Ar-H), 7.67 (s, 1H, Ar-H), 8.19 (d, 1H,
J = 7.9 Hz, Ar-H), 8.87 (s, 1H, Ar-H), 9.23 (s, 1H, Ar-H). 13 C-NMR (75 MHz
CDCl3) d: 24.1, 36.1, 40.9, 45.1, 59.1, 114.5, 123.1, 125.4, 128.5, 129.9,
131.5, 133.7, 138.6, 146.3, 157.3, 159.2, 161.9, 174.8. MS (70 eV) m/z
(%): 307 [M+] (94), 277 (79), 264 (88), 249 (47), 221 (25), 141 (34), 113
(59), 98 (31), 57 (39), 43 (92). Anal. calcd. for C18H17N3O2 (307.35): C,
70.35; H, 5.53; N, 13.68%; found, C, 70.37; H, 5.50; N, 13.65%.
Synthesis of ethyl-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine-4-carboxylate
(19a-b). The mixture of 2-aminoquinoline-3-carbaldehyde (1a-b; 0.001 mol)
and ethyl-2-oxocyclohexane-carboxylate (18; 0.001 mol) in ethanolic po-
tassium hydroxide solution (10 mL, 2%) was refluxed for 2.5 h. Completion
of the reaction was monitored by TLC. The mixture was then cooled to
room temperature; the separated solid product was collected by suction
filtration, washed with pet-ether, dried and recrystallized from ethanol.
Ethyl-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine-4-carboxylate (19a).
Yield: 0.244 g (79%), recrystallized from ethanol to afford a faint green solid;
m.p. 117–119 ꢀC. IR (KBr): 3009 m, 2961 w, 2914 m, 1732 s, 1623 s, 1024 s cm–1.
1H-NMR (300 MHz CDCl3) d: 1.30 (t, 3H, J=6.2 Hz, CH3), 1.78 (m, 2H, CH2), 2.25
(m, 2H, CH2), 2.61 (t, 2H, J=5.9 Hz, CH2), 3.93 (t, 1H, J=5.3 Hz, CH), 4.09 (q, 2H,
J=6.2 Hz, CH2), 7.51 (dd, 1H, J=8.2 and 8.7 Hz, Ar-H), 7.67 (dd, 1H, J=8.7 and
8.1 Hz, Ar-H), 8.15 (d, 1H, J=8.2 Hz, Ar-H), 8.30 (d, 1H, J=8.1 Hz, Ar-H), 8.77 (s,
1H, Ar-H), 9.23 (s, 1H, Ar-H). Anal. calcd. for C19H18N2O2 (306.36): C, 74.50; H,
5.88; N, 9.15%; found, C, 74.52; H, 5.94; N, 9.22%.
tert-butyl-3,4-dihydroquinolino[2,3-b][1,6]naphthyridine-2(1H)-carboxylate
(21e). Yield: 0.279 g (83%), recrystallized from ethanol to afford a faint
purple solid; m.p. 267–269 ꢀC. IR (KBr): 3007 m, 2987 m, 2938 m, 1732 s,
1617 s, 1019 s cm–1 1H-NMR (300 MHz CDCl3) d: 1.59 (s, 9H, CH3), 3.26
.
(t, 2H, J = 6.7 Hz, CH2), 3.37 (t, 2H, J = 6.7 Hz, CH2), 4.34 (s, 2H, CH2), 7.32
(dd, 1H, J = 8.2 and 8.5 Hz, Ar-H), 7.67 (dd, 1H, J = 8.5 and 8.1 Hz, Ar-H),
8.17 (d, 1H, J = 8.2 Hz, Ar-H), 8.30 (d, 1H, J = 8.1 Hz, Ar-H), 8.88 (s, 1H,
Ar-H), 9.24 (s, 1H, Ar-H). Anal. calcd. for C20H21N3O2 (335.40): C, 71.64;
H, 6.26; N, 12.53%; found, C, 71.65; H, 6.26; N, 12.53%.
Ethyl-9-methoxy-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine-4-carboxylate
(19b). Yield: 0.274 g (81%), recrystallized from ethanol to afford a green
solid; m.p. 81–83 ꢀC. IR (KBr): 3026 m, 2976 m, 2942 m, 1738 s, 1620 s,
1041 s cm–1
.
1H-NMR (300 MHz CDCl3) d: 1.31 (t, 3H, J= 5.9Hz, CH3), 1.66
tert-butyl-9-methoxy-3,4-dihydroquinolino[2,3-b][1,6]naphthyridine-2(1H)-
carboxylate (21f). Yield: 0.301 g (82%), recrystallized from ethanol to af-
ford a faint green solid; m.p. 234–237 ꢀC. IR (KBr): 3019 m, 2954 m,
(m, 2H, CH2), 2.21 (m, 2H, CH2), 2.59 (t, 2H, J = 6.5Hz, CH2), 3.79 (t, 1H,
J= 6.0Hz, CH), 4.02 (s, 3H, OCH3), 4.11 (q, 2H, J = 5.9Hz, CH2), 7.48 (d, 1H,
J= 8.2Hz, Ar-H), 7.59 (s, 1H, Ar-H), 8.28 (d, 1H, J = 8.2Hz, Ar-H), 8.92 (s,
1H, Ar-H), 9.33 (s, 1H, Ar-H). 13C-NMR (75 MHz CDCl3) d: 12.2, 22.7, 28.3,
35.3, 49.3, 58.2, 62.3, 116.3, 123.3, 124.3, 129.3, 131.3, 133.7, 135.2, 138.3,
145.3, 158.3, 159.4, 163.7, 177.3. Anal. calcd. for C20H20N2O3 (336.38): C,
71.42; H, 5.95; N, 8.33%; found, C, 71.40; H, 5.95; N, 8.30%.
1736 s, 1612 s, 1028 s cm–1 1H-NMR (300 MHz CDCl3) d: 1.48 (s, 9H, CH3),
.
3.27 (t, 2H, J = 5.9 Hz, CH2), 3.42 (t, 2H, J = 5.9 Hz, CH2), 4.07 (s, 3H, OCH3),
4.42 (s, 2H, CH2), 7.47 (d, 1H, J= 8.4Hz, Ar-H), 7.63 (s, 1H, Ar-H), 8.37 (d, 1H,
J= 8.4 Hz, Ar-H), 8.94 (s, 1H, Ar-H), 9.24 (s, 1H, Ar-H). 13 C-NMR (75 MHz
CDCl3) d: 25.7 (3 Â Cs), 31.7, 42.1, 45.3, 59.1, 82.7, 115.3, 123.2, 125.7,
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D. P. Shelar et al.
128.7, 129.9, 132.7, 135.1, 137.9, 145.2, 158.8, 159.1, 159.9, 161.2. MS (70 eV)
m/z (%): 365 [M+] (76), 308 (48), 277 (37), 264 (59), 236 (19), 221 (32), 153
(38), 77 (26), 57 (94), 44 (57), 41 (84). Anal. calcd. for C21H23N3O3 (365.43):
C, 69.04; H, 6.30; N, 11.50%; found, C, 69.04; H, 6.32; N, 11.52%.
the transformation of functional groups from the starting ketone
into the annelated heterocyclic ring. Thus, base-catalysed conden-
sation (in ethanolic KOH) of 1 with cyclopentanone 6a gave the
strained heterocycle 2,3-dihydro-1H-benzo[b]cyclopenta[g][1,8]
naphthyridine 7(a-b) in 80–84% yield, also available via acid
catalysed condensation (in aq. HCl) of the components although
in much lower yield, i.e. 40–45%, hence we performed further
cyclocondensation in the base-catalysed condensation. There-
fore, cyclocondensation of 2-aminoquinoline-3-carbaldehyde 1a
or 1b with 2-methylcyclohexanone 8 in ethanolic KOH furnished
into 4-methyl-1,2,3,4-tetrahydrodibenzo[b,g][1,8]naphthyridine 9
(a-b) in 82–85% yield. However, reaction of 1 with dimedone
10(a-b) was unsuccessful in ethanolic KOH due to its more enolic
character; hence this condensation is achieved by heating
without solvent at 180–185 ꢀC to afford 3,3-dimethyl-3,4-
dihydrodibenzo[b,g][1,8]naphthyridin-1(2H)-one 11(a-d) in good
yield (Scheme 2).
Numerous six-membered ring ketones have been converted
into fused system via their annelation with o-aminoaldehyde.
From the literature, we observe that a large number of substituted
piperidones (55,56) and pyrrolidones have been condensed with
o-aminobenzaldehyde in the base-catalysed reaction; thus, herein,
we utilized the piperidones for generating the various hetero-
cycles, by Friedländer condensation with 2-aminoquinoline-3-
carbaldehyde (o-aminoaldehyde) 1.
Because of useful applicability of base-catalysed condensation,
we performed further condensations in ethanolic KOH. Thus, con-
densation of 1a or 1b with cetralene 12 and 6-methoxy-tetralone 14
in ethanolic KOH under reflux gave 5-(3,4-dichloro-phenyl)-5,6-
dihydrobenzo[b]naphtho[2,1-g][1,8]naphthyridine 13(a-b) and
3-methoxy-5,6-dihydrobenzo[b]naphtho[2,1-g][1,8]naphthyridine
15(a-b), respectively, in good yield. Analogue cycloconden-
sation of 3,3-dimethyl-1,5-dioxaspiro[5,5]undecan-9-one 16,
ethyl-2-oxocyclohexane-carboxylate 18 and substituted piperidone
20(a-c) under similar reaction conditions yielded 5′,5′-dimethyl-3,4-
dihydro-1H-spiro[dibenzo[b,g]-[1,8]naphthyridine-2,2′-[1,3]dioxane]
17(a-b), ethyl-1,2,3,4,-tetrahydrodibenzo[b,g][1,8]-naphthyridine-4-
carboxylate 19(a-b), and 2-methyl-1,2,3,4-tetrahydroquinolino[2,
3-b][1,6]- naphthyridine 21(a-f), respectively, in good yield. All syn-
thesized compounds were characterized by spectroscopic and ana-
lytical methods and all spectra is provided in Supporting information
section. For example, the IR of 21d shows stretching frequency at
1683 cm–1 for C = O. The 1H-NMR spectrum of 21d in CDCl3 shows
three singlets at 2.09, 4.05 and 4.81 ppm corresponding to protons
of methyl, methoxy and methylene groups, respectively. Two triplets
at 2.78 and 3.19 ppm with J=6.4Hz observed for the –CH2–CH2–
group. All aromatic protons show expected chemical shifts and split-
ting patterns which resemble the structure proposed. The mass spec-
trum of 21d reveals a molecular ion peak m/z at 307. The 13C-NMR
spectrums of this compound are in agreement with the structure
proposed (Scheme 3). The above study revealed that base-catalysed
condensation is a more elegant method than other reaction condi-
tions reported in literature. Synthesized benzonaphthyridines were
further utilized for study of their photophysical properties.
Results and discussion
Annelation reaction with heterocyclic aminoaldehydes pro-
vides synthetic entry into heterocyclic systems fused to a pyri-
dine or pyrimidine (44–47) nucleus by condensation reaction.
o-Aminoaldehyde has fascinating potential, remarkable versa-
tility and utility for the annelation of heterocyclic ring
structures to synthesize various heterocycles (48–53). Known
2-aminoquinoline-3-carbaldehyde (o-aminoaldehyde) 1 has
been synthesized by a novel method reported in our previous
communication (42).
Friedländer synthesis was originally described by Friedländer
in 1882 and has been accepted to be one of the most convenient
routes to this scaffold (54). The reaction advances by the
condensation of a-amino carbonyl compound (N–C–C–C unit)
with a derivative having a-methylene group with respect to the
carbonyl (C–C unit), in the presence of a suitable catalyst. During
recent years there has been a rapid increase in the number of
publications describing the successful accomplishment of the
Friedländer reaction. This was achieved by making changes in
the catalyst or reaction medium or physical parameters, although
the starting substrates were conceptually similar in these endeav-
ours. From the literature, we observe that Friedländer condensa-
tion is generally carried out either with base catalysis (sodium or
potassium hydroxide, sodium alkoxide, piperidine) in water or
alcoholic solvents, or by heating a mixture of the components
in the absence of solvent, or heating in high-boiling solvent;
also, Friedländer condensation is carried out with acid catalysis
or with Lewis acid or in under microwave irradiation.
Hence, to determinate suitable reaction conditions for
Friedländer condensation, we performed cyclocondensation of
1a or 1b with formamide 2a in various reaction conditions,
such as: (i) in ethanolic KOH under reflux; (ii) neat heat about
175–180 ꢀC without solvent; (iii) reflux in high-boiling solvent,
i.e. in diphenyl ether for 2–3 h; (iv) in aq. HCl as strong acid at
90 ꢀC for 6 h; (v) heat with SnCl2. 2H2O (Lewis acid); (vi) use of
HCl as a catalyst for a similar reaction under microwave irradia-
tion for benzonaphthyridine synthesis (unlike the conventional
heating, which takes about 6 h for completion of the reaction,
microwave speeds up the reaction and completes it within
1.5–12 min). From the above studies, we observed that the
base-catalysed reaction, i.e. milder reaction conditions, gave
good yield and, more importantly, lack of side-products as com-
pared to other reaction conditions. Thus, cyclocondensation of
1a or 1b with amide 2(a-b) achieved in ethanolic KOH to fur-
nished pyrimido[4,5-b]quinoline 3(a-d) in 81–85% yield. On the
other hand, a mild base, such as piperidine, was sufficiently strong
for condensation of o-aminoaldehyde 1 with benzyl cyanide.
Hence, cyclocondensation of 1a or 1b with benzyl cyanide 4 in
ethanolic piperidine gave 3-phenylbenzo[b][1,8]naphthyridin-2-
amine 5(a-b) in 81–84% yield (Scheme 1).
Reactions of o-aminoaldehydes with cyclic ketones are espe-
cially valuable for the construction of polycondensed heterocy-
clic systems. Herein, we extend Friedländer condensation using
different cyclic ketones having a reactive methylene group to
generate libraries of new heterocycles. And mild reaction condi-
tions are employed in the Friedländer condensation to permit
Photophysical properties
Absorption and fluorescence studies in homogeneous media
(organic solvents) and microheterogeneous media (micelles)
and dioxane–water system. A variety of environmental
factors affect fluorescence emission, including interactions
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Interaction of benzo[b][1,8]naphthyridines with HSA and BSA
O
H2N R1
R
2(a-b)
N
i) - vi)
N
N
R1
3 (a-d)
O
3a; R= H, R1 = H
3b; R= OCH3, R1 = H
3c; R= H, R1 = Ar
R
H
NH2
N
1 (a-b)
3d; R= OCH3, R1 = Ar
CN
1a; R = H
1b; R = OCH3
R
4
NH2
N
N
EtOH/ Piperidine
reflux, 6h
5 (a-b)
i) EtOH/ KOH, reflux, 3h
ii) 175-180 °C, neat heat, 1h
iii) Diphenyl ether, reflux, 2.5h
v) SnCl . 2H O, Neat heat, 15-140 min. vi) HCl Cat., MW, 1.5-12min.
iv) HCl (1.0 eq.) H2O, 90 °C, 6h
2
2
Scheme 1. Synthesis of benzo[b][1,8]naphthyridines by condensation of o-aminoaldehyde with amides and benzylcyanide.
O
(CH2)n
R
6 (a-b)
(CH2)n
EtOH/ KOH,
reflux, 5h
N
N
7 (a-d)
7a; R = H, n = 1
7b; R = OCH3, n = 1
7c; R =H, n = 2
7d; R = OCH3, n = 2
O
O
R
R
R
H
8
EtOH/ KOH,
reflux, 6h
N
1 (a-b)
NH2
N
N
O
9 (a-b)
R1
O
O
R2
10 (a-b)
R1
R2
N
N
180-185 °C,
15 mins.
11 (a-d)
11a; R, R1, R2 = H,
11b; R=OCH3; R1, R2 = H,
11c; R=H; R1, R2 = CH3,
11d; R=OCH3; R1, R2 = CH3
Scheme 2. Synthesis of benzo[b][1,8]naphthyridines by condensation of o-aminoaldehyde with alicyclic ketones.
between the fluorophore and surrounding solvent molecules
the solvation shell of the solutes (57). Organic mixed solvents
are widely used as the mobile phase in liquid chromatography
and capillary electrophoresis as a reaction medium. Solvent
mixtures have improved physical properties, such as solvation
power, density, viscosity and refractive index, compared with
their neat solvents (58). When the solute is dissolved in a solvent,
the solvent exerts a definite influence on the solute. This influ-
ence depends on the nature of the solvent. This influence
reflects changes in the absorption and fluorescence spectrum
(59) and this phenomenon is known as solvatochromism.
Solvatochromism is used to describe the pounced change in
position that sometimes occurs in the intensity of an absorption
band, accompanying a change in the polarity of the medium.
The preferential solvation phenomenon that is the selective
(dictated by solvent polarity), other dissolved inorganic and or-
ganic compounds, temperature, pH and the localized concentra-
tion of the fluorescent species. The effect of these parameters
varies widely from one fluorophore to another, but the absorp-
tion and emission spectra, as well as quantum yields, can be
heavily influenced by environmental variables. In fact, the high
degree of sensitivity in fluorescence is primarily due to interac-
tions that occur in the local environment during the excited
state lifetime. Thus, in this paper, we mainly give emphasis on
to the study of effect of solvent on absorption and fluorescence
emission, because solvents play an important role in physical
and chemical processes. Solvent effects are related to the nature
and the extent of the solute–solvent interactions developed in
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D. P. Shelar et al.
Cl
Cl
R
R
N
N
N
N
13 (a-b)
15 (a-b)
O
OMe
O
EtOH/KOH,
reflux, 2.5h
EtOH/KOH,
reflux, 3h
MeO
14
12
Cl
Cl
O
R
H
N
NH2
1 (a-b)
O
O
O
O
O
O
O
EtOH/ KOH,
reflux, 6h
EtOH/ KOH,
reflux, 3h
EtOH/ KOH,
reflux, 2.5h
N
R'
18
16
20 (a-c)
O
R'
R
R
R
N
O
N
N
N
N
N
N
21 (a-f)
21a; R=H, R'=CH3,
17 (a-b)
19 (a-b)
O
O
21b; R=OCH3, R'=CH3,
21c; R=H, R'= -COCH3,
21d; R=OCH3, R'= -COCH3,
21e; R=H, R'= -COOC(CH3)3,
21f; R=OCH3, R'= -COOC(CH3)3
Scheme 3. Synthesis of benzo[b][1,8]naphthyridines by condensation of o-aminoaldehyde with alicyclic ketones.
enrichment of a certain solvent component in the solvation shell
of a given solute is of paramount importance in the interpreta-
tion of many physiochemical parameters measured in the mix-
tures (60). Hydrogen bonding plays an important role in the
study of preferential solvation and has been widely investigated,
because it is present in large variety of chemical, biochemical
and pharmacological events (61).
Table 3 and graphically represented in Figs 2 and 3 for 5b and
21f, respectively. From Table 3 we observe that, in 1,4-dioxane–
water mixtures the absorption maximum (labs max) tends to de-
crease as the water content is increased. These absorption spectral
features can be due to ground state hydrogen bond interactions
and aggregate formation, particularly in water, in which benzo-
naphthyridines are not freely soluble. On the other hand, fluores-
cence maximum (lf max) is highly red-shifted as the water content
in the solvent mixture is increased; also it was observed that en-
hancement in quantum yield (Фf) is linear with the increase in
the percentage of water in the solvent mixtures. This observation
is similar to those from studies in organic solvents of different
polarity.
In contrast to a rather moderate solvent polarity effect on the
absorption spectra, significant solvent polarity effects are seen in
the fluorescence behaviour of these benzonaphthyiridines. In
organic solvents and dioxane–water systems, as the polarity
(relative permittivity, e) of the medium is increased, the lf max
undergoes a red shift.
In micellar media, benzonaphthyridines 3d shows a fluores-
cence peak at around 505–508 nm, similar to its fluorescence
in polar solvents such as methanol and acetonitrile. The fluores-
cence spectrum of compounds 21f in micelles is significantly
red-shifted as compared to that in non-polar n-hexane. How-
ever, the fluorescence maxima (lf max) in micelles are similar
to those in polar solvents such as DMF, MeCN and MeOH. As
compared to in polar solvents, the fluorescence maximum (lf
max) of 21f is blue-shifted in Triton-X100 micelles (Table 2,
Fig. 4). The fluorescence intensity of these studied compounds
in CTAB micelles is high compared to the SDS and Triton-X100
micelles. In general, as compared to the fluorescence of benzo-
panaphthyridines 3d, 5b and 21f in non-polar n-hexane, in
Prompted by this literature survey, in this study we investi-
gated the electronic absorption and fluorescence properties of
some representative benzonaphthyridines (3d, 5b and 21f) in
the homogeneous media of organic solvents with different
solvent polarities, dioxane–water binary mixtures and in the
microheterogeneous media of SDS, CTAB and Triton-X100. UV-vis
absorption and fluorescence spectral data of the benzo-
naphthyridines 3d, 5b and 21f in organic solvents (homogeneous
media) of different polarities are given in Table 1. Representative
fluorescence spectra in different organic solvents are shown in Fig. 1.
A moderate red shift in the absorption labs max of these com-
pounds is observed, whereas the fluorescence maximum (lf max)
of these compounds is greatly affected by the solvent polarity,
where a much red-shifted fluorescence is observed on increasing
the solvent polarity from n-hexane to acetonitrile (Fig. 1). We found
that the fluorescence intensity of these compounds in non-polar
n-hexane is low as compared to polar aprotic and polar protic sol-
vents (Fig. 1).
As compared to the homogeneous media of organic solvents,
the micellar environment does not cause a significant change in
the absorption spectra of these benzonaphthyridines (Table 2).
Only a very moderate red shift in the absorption labs maxima
of compounds 3d and 21f is observed. The absorption and fluo-
rescence spectral data of benzonaphthyridines 3d, 5b and 21f in
various compositions of 1,4-dioxane–water are summarized in
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Interaction of benzo[b][1,8]naphthyridines with HSA and BSA
Table 1. UV-vis absorption (labs max) and fluorescence (lf max) spectral data of benzonaphthyridines 3 d, 5b and 21f in homo-
geneous media (organic solvents)
Compound
3d
Solvent
Maximal wavelength (nm)
Absorption Emission
377 460
Stokes’ shift (cm–1)
Фf (Æ 0.002)
n-Hexane
Dioxane
THF
3712
4452
5712
6109
7223
7118
4672
5125
6029
7548
8230
8470
3724
4210
4968
5670
5951
6170
0.252
0.317
0.343
0.427
0.419
0.412
0.322
0.359
0.363
0.438
0.429
0.423
0.222
0.256
0.316
0.366
0.339
0.335
381
385
394
389
388
382
387
390
398
393
392
360
367
371
378
375
374
475
486
512
507
505
472
487
492
525
514
512
448
465
473
495
484
482
DMF
MeCN
MeOH
n-Hexane
Dioxane
THF
5b
DMF
MeCN
MeOH
n-Hexane
Dioxane
THF
DMF
MeCN
MeOH
21f
Figure 1. UV-vis absorption (labs max) and fluorescence (lf max) spectra of 5b in: (i) n-hexane; (ii) dioxane; (iii) THF; (iv) methanol; (v) MeCN; and (vi) DMF.
micellar media the fluorescence of these benzonaphthyridines is
red-shifted; the maximum red shift is observed for 21f. A
comparison of the lf max of benzopanthyridines 3d, 5b and
21f in polar organic solvents such as methanol with those in
micelles reveals that the benzonaphthyridines experience rela-
tively polar environment in the micelles.
The quantum yield (Фf) of benzonaphthyridines (3d, 5b and
21f) in organic solvents is greatly dependent on solvent polarity.
The fluorescence efficiency of benzonaphthyridines 5b and 21f
is generally decreased in polar solvents. The quantum yield
(Фf) of these benzonaphthyridines (3d, 5b and 21f) is relatively
higher in Triton-X100 as compared to either SDS or CTAB. The
fluorescence efficiency is lowest in SDS micelles for all three
benzonaphthyridines. Since we have already shown that there
is an increase in quantum yield (Фf) with increase in solvent
polarity of the medium, the increase in Фf in micelles is attribut-
able to the greater polarity of the medium.
The Stokes’ shift for all three benzonaphthyridines in polar
homogeneous media of organic solvents such as DMF, acetoni-
trile and methanol is greater than that in micellar media. Among
the three micelles studied, the smallest Stokes’ shift is observed
in neutral Triton-X100. Thus, the ionic micelles of SDS and CTAB
affect the fluorescence spectra most. This can be due to interac-
tions between the micellar charges and the polar benzo-
naphthyridine fluorophore. Thus, while benzonaphthyridine
21f shows significant micelle-charge dependent fluorescence
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D. P. Shelar et al.
Table 2. UV-vis absorption (labs max) and fluorescence (lf max) spectral data of benzonaphthyridines 3d, 5b and 21f in microhe-
terogeneous media (micelles)
Compound
Media
Maximal wavelength (nm)
Absorption Emission
397 508
Stokes’ shift (cm–1)
Фf (Æ 0.002)
3d
5b
21f
CTAB
SDS
Triton-X-100
CTAB
SDS
Triton-X-100
CTAB
SDS
5992
5992
5746
7322
6177
6171
4839
5340
4187
0.419
0.212
0.437
0.391
0.242
0.437
0.378
0.312
0.412
397
397
395
392
395
386
381
386
508
505
519
504
511
489
490
469
Triton-X-100
Table 3. UV-vis absorption (labs max) and fluorescence (lf max) spectral data of 3d, 5b and 21f in 1,4-dioxane–water mixtures
Compound
3d
Water in 1,4-dioxane (%)
labs max (nm)
lf max (nm)
Stokes’ shift (cm–1)
Фf (Æ 0.002)
0
10
20
30
40
50
60
0
10
20
30
40
50
60
0
381
378
375
373
370
368
366
387
385
384
380
376
374
371
367
364
360
359
355
353
349
475
485
488
488
491
493
493
487
503
503
503
504
504
504
465
469
483
498
505
508
519
4452
5789
5610
5610
5475
5475
5701
5125
5852
5892
5891
5908
6102
6227
4210
4109
5127
6451
6869
7223
7610
0.310
0.297
0.287
0.287
0.289
0.290
0.290
0.359
0.317
0.310
0.310
0.312
0.312
0.316
0.256
0.247
0.230
0.212
0.192
0.187
0.172
5b
21f
10
20
30
40
50
60
Figure 2. UV-vis absorption and fluorescence emission (lf max) spectra of 5b in different concentrations of water: (i) 0%; (ii) 10%; (iii) 20%; (iv) 30%; (v) 40%; (vi) 50%; and
(vii) 60%.
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Interaction of benzo[b][1,8]naphthyridines with HSA and BSA
Figure 3. UV-vis absorption and fluorescence emission (lf max) spectra of 21f in different concentration of water: (i) 0%; (ii) 10%; (iii) 20%; (iv) 30%; (v) 40%; (vi) 50%; and
(vii) 60%.
Figure 4. Fluorescence spectra of all three benzonaphthyridines (3d, 5b and 21f) in micelles of: (a) CTAB; (b) SDS; and (c) Triton-X100.
peak shifts, 3d and 5b do not show such marked changes in
their fluorescence lf max. Further, UV-vis and fluorescence
spectra of compounds 3(a-d), 5(a-b), 7(a-d), 9(a-b), 11(a-d),
13(a-b), 15(a-b), 17(a-b), 19(a-b) and 21(a-f) are taken in DMF
as solvent. Fluorescence quantum yields of all the synthesized
compounds were determined by a standard literature procedure
using quinine sulphate as a reference standard (62,63) and are
given in Table 4.
Trp-212). This additional tryptophan residue in BSA is located
at position 134, buried in a hydrophobic pocket and it has been
proposed to lie near the surface of the albumin molecule in the
second helix of the first domain (2,4,5). The tryptophan residue
plays an important role as a chromophore and a fluorophore
in optical studies of proteins.
Recently, we bound some representative benzonaphthyri-
dines with BSA and studied the effect of protein–probe interac-
tion on electronic absorption and fluorescence emission (43). In
continuation of our efforts to study the effect of protein–probe
(benzonaphthyridine) interaction (in phosphate buffer) on fluo-
rescence emission, herein we study the binding effect of synthe-
sized benzonaphthyridines (3d, 5b, 21f) with human serum
albumin (HSA) and bovine serum albumin (BSA) on absorption
and fluorescence properties. In order to examine the interaction
of 3d, 5b and 21f with HSA and BSA, the fluorescence titrations
with HSA and BSA were carried out in phosphate buffer of pH
7.4. The absorption and fluorescence spectral data for 3d, 5b
and 21f are collected in Tables 5 and 6 and fluorescence
emission (lf max) is graphically represented in Figs 5 and 6 with
HSA and BSA, respectively. From Tables 5 and 6, we observed
Electronic absorption and fluorescence properties of human
serum albumin (HSA) and bovine serum albumin (BSA) with
compounds 3d, 5b and 21f in phosphate buffer
HSA and BSA are homologous proteins composed of single poly-
peptide chains with 583 and 585 amino acids, respectively, with
a similar sequence and a similar conformation (64). From the
spectroscopic point of view, one of the main differences
between the two proteins is that in HSA there is only one trypto-
phan residue, which is located at position 214 (Trp-214), buried
in a hydrophobic pocket at sub-domain IIA, whereas in BSA
there are two tryptophan amino acid residues (Trp-134 and
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D. P. Shelar et al.
Table 4. UV-vis absorption (labs max) and fluorescence (lf max) spectral data of 3(a–d), 5(a–b), 7(a–d), 9(a–b), 11(a–d), 13(a–b), 15
(a–b), 17(a–b), 19(a–b) and 21(a–f) in DMF as the solvent (ca. 10–3) at room temperature
Compound
labs max (nm)
lf max (nm)
fF (Æ 0.002)
Compound
labs max (nm)
lf max (nm)
fF (Æ 0.002)
3a
3b
3c
3d
5a
5b
7a
7b
7c
7d
9a
9b
11a
11b
11c
335
361
365
394
365
398
360
365
362
369
365
368
368
373
370
451
467
461
512
474
525
472
481
477
488
472
481
478
498
489
0.256
0.294
0.284
0.427
0.315
0.438
0.294
0.327
0.301
0.345
0.284
0.378
0.311
0.397
0.350
11d
13a
13b
15a
15b
17a
17b
19a
19b
21a
21b
21c
21d
21e
21f
407
398
418
370
407
366
370
397
402
361
368
334
338
360
378
500
472
487
479
507
466
471
489
521
482
494
449
458
445
495
0.403
0.351
0.372
0.337
0.392
0.310
0.329
0.387
0.419
0.367
0.388
0.290
0.312
0.281
0.366
Table 5. Absorption maximum (labs max), fluorescence emission (lf max) and fluorescence quantum yield (Φf) of benzonaphthyr-
idines (3d, 5b and 21f) in phosphate buffer, pH 7.4, and HSA (5.0 Â 10–6 mol/L)
Compound
labs max (nm)
lf max (nm)
fF (Æ 0.002)
Buffer
HSA
Buffer
HSA
Buffer
HSA
3d
5b
21f
407
390
401
403
387
399
512
488
502
529
494
517
0.421
0.382
0.409
0.433
0.405
0.425
Table 6. Absorption maximum (labs max), fluorescence emission (lf max) and fluorescence quantum yield (Φf) of benzonaphthyr-
idines (3d, 5b and 21f) in phosphate buffer, pH 7.4, and BSA (5.0 Â 10–6 mol/L)
Compound
labs max (nm)
lf max (nm)
fF (Æ 0.002)
Buffer
BSA
Buffer
BSA
Buffer
BSA
3d
5b
21f
415
427
374
409
406
375
509
521
502
535
551
479
0.419
0.428
0.407
0.437
0.448
0.413
Figure 5. Comparative fluorescence emission (lf max) spectra of 3d, 5b and 21f: (A) phosphate buffer; (B) in HSA.
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Interaction of benzo[b][1,8]naphthyridines with HSA and BSA
Figure 6. Comparative fluorescence emission (lf max) spectra of 3d, 5b and 21f: (A) phosphate buffer; (B) in BSA.
that binding with BSA and HSA in the solutions of benzo-
naphthyridines in phosphate buffer results in an enhancement
of the fluorescence emission (red-shift) and quantum yield (Φf)
for all compounds, and blue shift in the lf max is observed for
5b with HSA and 21f with BSA. The blue shift in lf max suggests
that the polarities of the protein environments in which the
benzonaphthyridines are located are less than the polarity of
the bulk aqueous phase, since similar blue shifts are observed
in less polar organic solvents. This proves the binding of the
probes to a hydrophobic site of the protein. Further, we exam-
ined the effect of increasing HSA and BSA concentration on
fluorescence emission and noted that a gradual increase in the
concentration of HSA and BSA in the solution of benzonaphthyr-
idines (3d, 5b and 21f) in phosphate buffer results in an
enhancement of the fluorescence intensity and quantum yield
(Φf) for all three benzonaphthyridines. Representative fluores-
cence spectra in different concentration of HSA and BSA for 5b
and 21f are shown in Figs 7 and 8, respectively.
different concentrations of 3d, 5b and 21f are recorded in the
range 300–500 nm by exciting the protein at 280 nm. The repre-
sentative fluorescence emission spectra are given in Fig. 9. The
fluorescence intensity of BSA decreased regularly with the
increasing concentration of the probes, which indicates that
the probes are binding to the protein. Upon addition of increas-
ing amounts of compounds 3d, 5b and 21f, the fluorescence
intensity of BSA at 345 nm decreased, along with the appear-
ance of new peaks at 505, 512 and 497 nm, respectively.
Conclusions
In conclusion, the formation of linear tetracyclic and pentacyclic
benzonaphthyridines from dicyclic o-aminoaldehyde 1 repre-
sents a remarkable efficient heteroannelation reaction. All these
synthesized compounds are included in the library of fluores-
cent heterocyclic compounds. Synthesized benzonaphthyridines
were studied for their photophysical properties and certain
benzonaphthyridines (3d, 5b and 21f) were studied for UV-vis
absorption (labs max) and fluorescence emission (lf max) in
homogeneous media of organic solvents, 1,4-dioxane–water
binary mixtures and in the micellar environment. From these
studies, we reveal that, these benzonaphthyridines (3d, 5b and
21f) exhibit solvatochromic fluorescence emission that may be
Fluorescence quenching study with BSA
The interaction of benzonaphthyridines (3d, 5b and 21f) with
BSA was also monitored by studying the quenching of BSA fluo-
rescence with the increasing concentration of benzonaphthyri-
dines. Thus, the fluorescence spectra of BSA in the presence of
Figure 7. Fluorescence emission (lf max) spectra of 5b and 21f with increasing HSA concentration (A) for 5b; curves a–f correspond to [HSA] 0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 (Â
10–6 mol/L), respectively. (B) For 21f; curves a–f correspond to [HSA] 0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 (Â 10–6 mol/L), respectively.
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D. P. Shelar et al.
Figure 8. Fluorescence emission (lf max) spectra of 5b and 21f with increasing BSA concentration: (A) for 5b; curves a–f correspond to [BSA] 0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 (Â
10–6 mol/L), respectively; (B) for 21f; curves a–f correspond to [BSA] 0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 (Â 10–6 mol/L), respectively.
Figure 9. Fluorescence emission (lf max) spectra of BSA (1.0 Â 10–6 mol/L) in the presence of different concentrations of 5b and 21f: (A) for 5b, curves a–f correspond to
0.0, 0.4, 0.8, 1.2, 1.6 and 2.0 (Â 10–6 mol/L); (B) for 21f, curves a–f correspond to 0.0, 0.2, 0.4, 0.6, 1.2 and 1.6 (Â 10–6 mol/L).
due to a polar, conformationally relaxed, intramolecular charge-
transfer excited state. The absorption maximum (labs max) of these
compounds undergoes a moderate red shift with increasing sol-
vent polarity; on the other hand, the fluorescence maximum
(lf max) becomes highly red-shifted with increasing solvent
polarity. However, these benzonaphthyridines (3d, 5b and 21f)
exhibit micellar charge-dependent changes in their fluorescence in-
tensity in the micellar environment. These fluorescence properties
of benzonaphthyridines can be utilized to investigate the micro-
environment of ionic micelles and related organized assemblies.
Further, fluorescence probe properties of these benzonaphthyr-
idines (3d, 5b and 21f) with HSA and BSA were investigated using
UV-vis absorption and fluorescence spectroscopy. Upon binding
with HSA and BSA, these compounds exhibit enhanced fluores-
cence intensity and quantum yield (Φf), except for 5b with HSA
and 21f with BSA, which show blue-shifted fluorescence intensity.
In general, fluorescence intensity enhancement is linear with the
increase in HSA and BSA concentration. The results indicate that
these benzonaphthyridines can be used for studying the polarities
of protein cavities. This study provides new directions for the
design of neutral and hydrophobic fluorescence probes. In
general, these compounds have good potential for use as fluores-
cence probes to study the microenvironments of biomolecules,
polymers, organized assemblies, etc. Quantum yields (Φf) of all
synthesized compounds are calculated.
SUPPORTING INFORMATION
The supporting information i.e. 1H and 13C NMR, mass spectra
may be found in the online version of this article.
Acknowledgements
The authors would like to thank the Council of Scientific and
Industrial Research (CSIR), New Delhi, India, for financial support
for this research project. They thank Professor D. D. Dhavale,
Department of Chemistry, University of Pune, India, for his
1
valuable cooperation in the measurement of H-NMR, 13C-NMR,
mass and elemental analysis. We thank Dr V. B. Gaikwad, Principal,
KRT Arts, BH Commerce and AM Science College, Nashik-02, MS,
India, for valuable cooperation in the measurement of IR, UV
and fluorescence.
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Interaction of benzo[b][1,8]naphthyridines with HSA and BSA
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