Synthesis of 1,8-naphthyridines and their application in the development of anionic fluorogenic chemosensors

Article abstract of DOI:10.1007/s10895-012-1041-5

Two 1,8-naphthyridines were synthesized and found to be fluorescent in solution. These compounds were studied in the presence of Cu⁺ and Cu²⁺ ions and it was verified that the metal causes the quenching of their fluorescence emission

Full text of DOI:10.1007/s10895-012-1041-5

J Fluoresc (2012) 22:1033–1046
DOI 10.1007/s10895-012-1041-5
ORIGINAL PAPER
Synthesis of 1,8–Naphthyridines and Their Application
in the Development of Anionic Fluorogenic Chemosensors
Celso R. Nicoleti & Diogo N. Garcia & Luiz E. da Silva &
Iêda M. Begnini & Ricardo A. Rebelo &
Antonio C. Joussef & Vanderlei G. Machado
Received: 28 October 2011 /Accepted: 7 March 2012 /Published online: 27 March 2012
#
Springer Science+Business Media, LLC 2012
Abstract Two 1,8–naphthyridines were synthesized and
found to be fluorescent in solution. These compounds were
studied in the presence of Cu+ and Cu2+ ions and it was
verified that the metal causes the quenching of their fluo-
rescence emission, due to the formation of complexes be-
tween the naphthyridine and the metal. A displacement
assay was carried out in a DMSO–water mixture with the
addition of various anions to the solutions of the complexes,
and it was observed that these systems have a high capacity
to selectively detect cyanide.
these species are of fundamental importance in many chem-
ical and biological processes. Therefore, many methodologies
based on optical chemosensors have been studied to perform
selective naked–eye and quantitative detection of anionic
species [1–17]. Of these methodologies, a strategy that has
been applied for the detection of anionic analytes involves the
concept of displacement assays [8–17]. In this strategy, a
compound especially tailored to act as a receptor for a given
analyte interacts with an indicator (a signalizing unit) forming
a complex, which causes an optical change in the system. The
addition of a particular analyte causes a competition scenario,
which results in the displacement of the indicator from its
initial complex, causing a perceptible change in the optical
signal of the system. Various displacement assays for the
detection of anions based on chromogenic and fluorogenic
units have been reported in the literature [8–17].
.
.
Keywords Displacement assays Copper Anion
.
.
.
sensing Cyanide 1,8–naphthyridines Fluorogenic
chemosensors
Of the anionic species to be recognized and detected,
CN– has attracted considerable interest because it has many
applications in metallurgy, fishing, mining, and the fabrica-
tion of polymers [18, 19]. This anion is lethal in very small
concentrations due to its ability to bind strongly to the active
site of cytochrome–oxidase, which leads to the inhibition of
the mitochondrial electron transport chain, and consequently
to a decrease in the oxidative metabolismo [18, 19]. CN– is
released through hydrolysis from some fruit seeds and roots
[20–22]. The chemical warfare agent known as tabun, deliv-
ers CN– through hydrolysis and this feature may be of
importance in the development of chemosensors for the
detection of this neurotoxic compound [23, 24]. Therefore,
many chemosensors for the detection of CN– have been
studied in the recent years [20–22, 25–35].
Introduction
The recognition and detection of anions [1–7] is a field that
has recently attracted increasing interest due to the fact that
:
:
:
C. R. Nicoleti D. N. Garcia I. M. Begnini R. A. Rebelo
Departamento de Química,
Universidade Regional de Blumenau, FURB,
Blumenau, SC 89010-971, Brazil
:
:
:
C. R. Nicoleti L. E. da Silva A. C. Joussef V. G. Machado (*)
Departamento de Química,
Universidade Federal de Santa Catarina, UFSC,
CP 476,
Florianópolis, SC 88040-900, Brazil
e-mail: vanderlei.machado@ufsc.br
Present Address:
L. E. da Silva
Universidade Federal do Paraná, UFPR, Setor Litoral,
rua Jaguariaíva, 512, Caiobá,
Matinhos, PR 83260-000, Brazil
1,8–Naphthyridine and its derivatives [36] are com-
pounds that have attracted considerable interest in recent years
because they exhibit various types of biological activity,
which include their action as chemotherapeutic and anti–

1034
J Fluoresc (2012) 22:1033–1046
infective agents, growth regulators, fungicides, nematocides,
and insecticides [37]. 1,8–Naphthyridine can also act as a
bidentate ligand, using the lone electron pairs of the nitrogen
atoms, leading to the formation of metal complexes [38].
Moreover, some of these compounds or their complexes dis-
play interesting photophysical properties [39–43], and there-
fore have the potential to function as fluorogenic chemosensors
for the detection of metal ions [42, 44–47]. Thus, the impor-
tance of these compounds has led to the development of many
synthetic methods aiming at their preparation and the study of
their applications [37, 38, 48–51]. It is important to note that
although 1,8–naphthyridines have been applied as fluorogenic
chemosensors for metal ions [42, 44–47], no literature refer-
ence on the use of these compounds to detect simple anionic
species is available.
(0.111 % water), and CH3COO− (0.067 % water). Buffered
solutions were prepared with 2–amino–2–hydroxymethyl–
propan–1,3–diol (tris, Sigma–Aldrich). 2,6–Diaminopyridine
(Sigma–Aldrich) was recrystallized from trichloromethane
before use. Meldrum’s acid was prepared by a procedure de-
scribed in the literature [53], through a reaction of malonic acid
in acetic anhydride with acetone in the presence of sulfuric acid.
Tetrakis(triphenylphosphine)palladium (0), [Pd(PPh3)4], with
purity of 99 %, was purchased from Sigma–Aldrich.
CuBF4.4CH3CN was prepared by reaction of Cu(BF4)2 with
copper in powder in anhydrous acetonitrile, through a procedure
reported in the literature [54].
The melting points were obtained on a Kofler hot stage and
were uncorrected. UV–vis measurements were performed with
a Varian Cary Bio 50 spectrophotometer and the emission
spectra were obtained with a Shimadzu RF–5301PC spectro-
fluorimeter, both equipped with thermostatted cell compart-
ments at 0.1 °C, using 1 cm quartz square cuvettes closed
with rubber septums to avoid the evaporation of the solvent.
The maxima of the UV–vis spectra (lmax) were calculated from
the first derivative of the absorption spectrum. The pH values
were determined with a Gehaka model PG 2000 pH meter. The
NMR spectra were recorded on Brucker AC–300 and Varian
AS–400 spectrometers. Chemical shifts were recorded in ppm
with the solvent resonance as the internal standard. Data are
reported as follows: chemical shift, multiplicity (s 0 singlet,
d 0 doublet, t 0 triplet), coupling constants (Hz) and integra-
tion. IR spectra were obtained on a Shimadzu model
Prestige–21 spectrometer, with KBr pellets. Microanaly-
sis was performed by Central de Análises–UFSC, using a
CHNOS elemental analyzer model EA 1110 CHNS–O from
CE Instruments.
In this study, the 1,8–naphthyridines 1 and 2 were syn-
thesized and characterized. Their photophysical properties
were investigated and these compounds were then used in
studies to evaluate their potential as selective fluorogenic
chemosensors for CN–, in a dimethyl sulfoxide (DMSO)–
water mixture, by means of a displacement assay.
Experimental
Materials and Methods
All chemicals used were high–purity commercial reagents.
DMSO was purified according to a procedure described in
the literature and then stored over molecular sieves (4 Å,
Sigma–Aldrich) [52]. Karl–Fischer titrations were per-
formed with this solvent and demonstrated the presence of
water in a concentration of 5.1×10−3 mol dm−3 (0.04 %
water). Acetonitrile (HPLC grade, Sigma–Aldrich) was
dried with calcium hydride (Sigma–Aldrich), distilled and
stored over 4 Å molecular sieves (Sigma–Aldrich), accord-
ing to the literature [52]. Deionized water was used in all
measurements. This solvent was boiled and bubbled with nitro-
gen and kept under a nitrogen atmosphere to avoid the presence
of carbon dioxide. Tetrahydrofurane (THF) was distilled from
sodium using benzophenone as an indicator [52]. Dichlorome-
thane was distilled from calcium hydride [52]. All other solvents
employed in the reactions were used as received. All anions
Synthesis and Characterization
N–(6–Amino–2–pyridinyl)acetamide (3) [55] A solution of
2,6–diaminopyridine (19 g; 174 mmol) and triethylamine
(20.5 cm3; 148 mmol) was prepared in THF (230 cm3). A
solution of acetic anhydride (14.1 cm3; 148 mmol) in
THF (60 cm3) was then added slowly, drop by drop,
under stirring. The reactional mixture was stirred for an
additional 14 h. Water (260 cm3) was then added to the
mixture and the THF was rotary–evaporated. The mixture
was left in a freezer overnight, and the white crystals
formed were filtered and dried under vacuum without
heating. Yield: 11.9 g (45 %); mp: 157–159 °C. IR
(KBr) νmax/cm−1: 3458 (ν N–H), 3358 (ν N–H), 3224
(ν N–H), 1672 (ν C0O), 1647, 1631, 1618, 1562, 1541,
1369, 1303, 1261, 1242, 1166, 989, 792. 1H–NMR
(300 MHz, CDCl3): δ 2.02 (s, 3H, −CH3); 5.69 (s, 2H, −NH2);
6.16 (d, J09 Hz, 1H, CHAr); 7.20 (d, J09 Hz, 1H, CHAr); 7.32
(t, J06 Hz, 1H, CHAr); 9.83 (s, 1H, −NH). 13C–NMR (75 MHz,
CDCl3): δ 23.92 (−CH3); 100.79; 103.21; 138.80; 150.50;
(HSO4 , H2PO4 , NO3 , CN−, CH3COO−, F−, Cl−, Br−, and I−)
were used as tetra–n–butylammonium salts with purity greater
than 97–99 %. The anions were purchased from Fluka
−
−
−
(F−, >97 %; Cl−, >98 %; NO3 , >97 %; and H2PO4 , >97 %),
−
−
Vetec (Br−, >99 %; I−, >99 %; and HSO4 , >99 %) and Sigma–
−
Aldrich (CH3COO−, >97 %). They were dried over phospho-
rous pentoxide under vacuum before use. Karl–Fischer
experiments were performed for the following tetra–n–butylam-
monium salts in order to determine the content of water in each
salt: CN− (0.116 % water), F− (1.125 % water), H2PO4
−

J Fluoresc (2012) 22:1033–1046
1035
158.42; 168.79 (−C0O). Anal. calcd. for C7H9N3O: C, 55.62; H,
6.00; N, 27.80. Found: C, 55.47; H, 6.02; N, 27.80.
(KBr) νmax/cm−1: 3450 (ν N–H), 2920 (ν C–H), 1697 (ν
C0O), 1601, 1537, 1489, 1435, 1393, 1306, 1240, 1134 (ν
1
C–Cl), 851, 827. H–NMR (300 MHz, CDCl3): δ 2.31
N–[6–[[(2,2–Dimethyl–4,6–dioxo–1,3–dioxan–5–ylidene)
methyl]amino]–2–pyridinyl] acetamide (4) Meldrum’s acid
(17.03 g; 118 mmol) was refluxed with trimethyl orthofor-
mate (71.5 cm3) for 2 h [56]. After this period compound 3
(8 g; 53 mmol) was added and the reflux was applied for a
further 20 min. The reactional mixture was cooled a little,
only to form the crystals, and the still hot mixture was
filtered using a Büchner funnel, the crystals being washed
with cold ethanol. The solid was dried under vacuum, yield-
(s, 3H, −CH3); 7.48 (d, J03 Hz, 1H, CHAr); 8.59 (d,
J09 Hz, 1H, CHAr); 8.65 (d, J09 Hz, 1H, CHAr); 8.88
(d, J06 Hz, 1H, CHAr); 9.47 (s, 1H, −NH). 13C–NMR
(75 MHz, CDCl3): δ 24.99 (−CH3); 116.24; 119.34;
120.84; 136.26; 142.85; 153.16; 154.61; 155.39;
169.86 (−C0O).
N–[5–(4–Methoxyphenyl)–1,8–naphthyridin–2–yl]acet-
amide (2) Compound 6 (0.4 g; 1.806 mmol), 4–methoxy-
phenylboronic acid (0.329 g; 2.167 mmol), and Pd(PPh3)4
(0.104 g; 0.09 mmol) were suspended in benzene (4.5 cm3),
ethanol (0.6 cm3), and Na2CO3 (2 mol dm−3; 1.6 cm3;
3.25 mmol). This mixture was refluxed (80 °C) for 9 h
and, after this time, more 4–methoxyphenylboronic acid
(0.055 g; 0.361 mmol) was added and the reflux was main-
tained for a further 5 h. The suspension formed was washed
with water (3×20 cm3) and extracted with dichloromethane
(3×30 cm3). The organic phase was preserved and the
aqueous phase was also washed with dichloromethane (2×
60 cm3). The organic phases were combined to be later
washed with water (3×75 cm3), dried with anhydrous
MgSO4, and rotary–evaporated. The dark–colored solid
was purified through flash column chromatography using
silica gel (60 G, 5–40 μm) as the adsorbent. The elution was
initiated with acetonitrile/ethyl acetate (1:1) and the polarity
was gradually increased during the elution using acetoni-
trile:ethyl acetate:etanol (4:4:1). The eluate was dried with
MgSO4 and rotary–evaporated. The product obtained was
dried, yielding 286 mg (54 %). mp 256–257 °C. IR (KBr)
ing 13.25 g (82 %). mp: 228.9–230.0 °C. IR (KBr) νmax
/
cm−1: 3255 (ν N–H), 1734 (ν C0O), 1691 (ν C0O), 1668
(ν C0O), 1620, 1570, 1544, 1444, 1394, 1313, 1276, 1255,
1244, 1190, 933, 804, 786. 1H–NMR (300 MHz, CDCl3): δ
1.76 (s, 6H, −CH3); 2.28 (s, 3H, −CH3); 6.73 (d, J09 Hz,
1H, CHAr); 7.74 (t, J06 Hz, 1H, CHAr); 8.06 (d, J09 Hz,
1H, CHAr); 8.10 (s, 1H, −NH); 9.27 (d, J015 Hz, 1H,
CHAr); 11.18 (d, J015 Hz, 1H, −NH). 13 C–NMR
(75 MHz, CDCl3): δ 24.70 (−CH3); 27.09 (−CH3); 88.65;
105.29; 107.89; 110.95; 141.40; 147.32; 150.64; 151.16;
163.36 (−C0O); 165.35 (−C0O); 168.87 (−C0O). Anal.
calcd. for C14H15N3O5: C, 56.56; H, 5.05; N, 14.14. Found:
C, 56.08; H, 5.06; N, 13.97.
N–(5,8–Dihydro–5–oxo–1,8–naphthyridin–2–yl)acetamide (5)
Diphenyl ether (200 cm3) was refluxed at 250 °C to add
slowly the Meldrum’s acid adduct 4 (5 g; 16.4 mmol). The
reflux was applied for a further 15 min. After cooling, ethyl
ether (80 cm3) was added. The suspension was filtered using
a Büchner funnel and washed with ethyl ether. The solid
obtained was washed with hot hexane, yielding 2.17 g
(84 %). mp: >300 °C (lit. [57] 310–315 °C). IR (KBr)
ν
max/cm−1: 3420 (ν N–H), 3285 (ν C–H, aromatic), 2930 (ν
C–H, aliphatic), 1701 (ν C0O), 1607, 1584, 1516, 1497,
1422, 1395, 1304, 1283, 1254 (ν C–O–C, asymmetric),
ν
max/cm−1: 3332 (ν N–H), 2953 (ν C–H), 1676 (ν C0O),
1612 (ν C0O), 1508, 1307, 1192, 815, 599. 1H–NMR
(300 MHz, DMSO): δ 2.14 (s, 3H, −CH3); 6.02
(d, J07.6 Hz, 1H, CHAr); 7.80 (d, J07.6 Hz, 1H, CHAr);
8.03 (d, J08.8 Hz, 1H, CHAr); 8.37 (d, J08.4 Hz, 1H,
CHAr); 10.69 (s, 1H, −NH); 11.64 (d, J04.8 Hz, 1H,
−NH). 13C–NMR (75 MHz, DMSO): δ 24.91 (−CH3);
110.50; 111.09, 117.53; 137.68; 140.47; 150.20; 154.78;
170.76 (−C0O); 177.59 (−C0O).
1233, 1180, 1026, 835. H–NMR (400 MHz, CDCl3): δ
1
2.30 (s, 3H, −CH3); 3.91 (s, 3H, −O–CH3); 7.08 (d,
J08.0 Hz, 2H, CHAr); 7.33 (d, J04.0 Hz, 1H, CHAr);
7.44 (d, J08.0 Hz, 2H, CHAr); 8.36 (d, J09.2 Hz, 1H,
CHAr); 8.47 (d, J09.2 Hz, 1H, CHAr); 9.00 (d, J04.0 Hz,
1H, CHAr); 9.35 (s, 1H, −NH). 13C–NMR (100 MHz,
CDCl3): δ 24.97 (−CH3); 55.46 (−O–CH3); 114.36;
115.34; 119.26; 120.79; 129.08; 130.84; 138.03; 149.42;
153.14; 153.78; 155.32; 160.30; 170.02 (−C0O). Anal.
calcd. for C17H15N3O2: C, 69.61; H, 5.15; N, 14.33. Found:
C, 69.48; H, 5.19; N, 14.24.
N–(5–Chloro–1,8–naphthyridin–2–yl)acetamide (6) [58,
59] POCl3 (18 cm3) and compound 5 (1 g; 4.93 mmol)
were heated at 90–95 °C for 1.5 h. The mixture was cooled
and ice was added under vigorous stirring. NH4OH was
added to the mixture until pH 8, the solid was filtered with
a Büchner funnel, and washed with ice cold water. The solid
was dried under vacuum, yielding 0.69 g (63 %; lit. [58]
79.7 %; lit. [59] 32–37 %). mp: >250 °C. (lit. [58] 265–
266 °C; lit. [59] 240 °C with previous decomposition). IR
5–(4–Methoxyphenyl)–1,8–naphthyridin–2–amine (1) A
suspension of 2 (0.5 mmol; 150 mg) in H2SO4 (10 %;
2 cm3) was heated and after solubilization was refluxed for
5 min. The pH of the reactional mixture was increased until
9 and the pale–yellow precipitate obtained was filtered in a
Büchner funnel and dried under vacuum at 60 °C to yield

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J Fluoresc (2012) 22:1033–1046
110 mg (85 %). mp: 243.6–246.2 °C (lit. [60] 233–234 °C).
IR (KBr) νmax/cm−1: 3456 (ν N–H), 3302 (ν N–H), 3157 (ν
C–H, aromatic), 1635, 1606, 1562, 1502, 1431, 1404, 1244,
1177, 1028, 822, 555. 1H–NMR (300 MHz, DMSO): δ 3.83 (s,
3H, −CH3); 6.80 (d, J09.6 Hz, 1H, CHAr); 6.83 (s, 2H, −NH2);
7.05 (d, J04.5 Hz, 1H, CHAr); 7.09 (d, J08.4 Hz, 2H, CHAr);
7.41 (d, J08.4 Hz, 2H, CHAr); 7.85 (d, J09.0 Hz, 1H, CHAr);
8.66 (d, J04.5 Hz, 1H, CHAr). 13C–NMR (75 MHz, CDCl3): δ
55.26 (−CH3); 113.35; 114.20; 114.48; 117.45; 129.42;
130.64; 135.10; 147.85; 151.34; 157.26; 159.49; 160.39. Anal.
calcd. for C15H13N3O: C, 71.70; H, 5.21; N, 16.72. Found: C,
71.47; H, 5.25; N, 16.65.
Compounds 1 and 2 as Anionic Fluorogenic Chemosensors
Two solutions of 1 and 2 (c01×10−6 mol dm−3) in DMSO–
water (7:3 v/v; tris/HCl, c01×10−3 mol dm−3; pH 8.5) were
prepared and then used in two experiments, in the selectivity
assay with the anions and for the titration of the complexes
involving Cu2+ and the naphthyridines with CN−. The selec-
tivity assay was performed obtaining the fluorescence emission
spectra at 25 °C for the solutions of the free naphthyridines, and
the fluorescence emission intensity (IF) was collected for their
maxima at the lem values (lem0408 nm for 1 and lem0440 nm
for 2). A solution of the complexes 1:Cu2+ and 2:Cu2+ was then
prepared, with Cu2+ in a concentration of 4.0×10−3 mol dm−3.
The solution of each complex was used to prepare 2 cm3 of
Photophysical Properties of 1 and 2
each solution of the anion (NO3 , CN−, F−, Cl−, Br−, I−, HSO4 ,
−
−
−
Buffered aqueous solutions were prepared using tris in a
concentration of 1×10−3 mol dm−3, the pH being adjusted to
8.5 and 6.0 with a solution of HCl 0.1 mol dm−3. Subse-
quently, solutions of 1 and 2 were prepared in the respective
buffers and in DMSO, in a concentration of 1×10−5 mol dm−3.
From these solutions, UV–vis spectra were obtained at 25 °C
and the absorbance values were collected at the maximum
wavelength for 1, at lmax0330 nm for water (pH 8.5) and
and H2PO4 ) in a concentration of 8×10−3 mol dm−3. The
fluorescence emission spectra were recorded for each solution
and the IF values were collected as previously described.
For the titrations with CN−, solutions (6 cm3) of 1:Cu2+
and 2:Cu2+ were performed with the solutions of 1 and 2,
with the same concentrations as those used in the selectivity
assays. From the solution of the complex, 1 cm3 was used to
prepare a stock solution of CN− (c06×10−2 mol dm−3) and
1.5 cm3 was placed in a quartz cuvette closed with a rubber
septum. After being thermostatized at 25 °C, a spectrofluori-
metric reading was taken. The titration was performed by
adding small aliquots of the solution of the anion with a
microsyringe. After each addition a spectrum was obtained,
the IF values being collected as shown in the preceding
paragraph. This procedure was followed until the return to
the IF value obtained for the free naphthyridine in solution.
A similar procedure was employed for the assays using Cu+,
but the concentration of the metal ion was 3×10−3 mol dm−3
and the concentration of the anions was 3×10−3 mol dm−3.
The CN− residues were treated by adding 5 mL of 10 %
NaOH (2.5 mol dm−3) and 70 cm3 of household bleach for each
50 mL of solution of CN− in the concentration of 2 % (w/v).
l
max0350 nm for DMSO, while for 2 the values were
obtained for lmax0321 nm (pH 6.0) and lmax0327 nm in
DMSO.
Samples were excited at the lmax values for 1 and 2 at
25 °C, with excitation and emission slit width settings of 5.0
and 1.5 nm, respectively. The quantum yields (Фf) were
determined for the dye using quinine sulfate (Фf,ref00.577
em H2SO4 0.1 mol dm−3) as an internal standard, with the
use of the expression Фf0Фf,ref (Aref/A)(η/ηref)(a/aref), A
being the absorbance of the sample, Aref the absorbance of
the internal standard, η and ηref the refractive index of the
solvent and the internal standard and a and aref the area
under the fluorescence peak of the sample and the internal
standard, respectively [61, 62].
pKa Values for 1 and 2 in Aqueous Solution
Calculations
Solutions of 1 and 2 were prepared in a concentration of
4×10−3 mol dm−3 in CHCl3, and stored in glass flasks
closed with rubber stoppers to avoid the evaporation of the
solvent. An aliquot of the solution, sufficient to give a
concentration of the compound of 1×10−5 mol dm−3 was
collected with a microsyringe and placed in a flask. After the
evaporation of the solvent, water at pH 2.22 (adjusted with
HCl 0.1 mol dm−3) was added and the fluorescence emission
spectrum of the naphthyridine was recorded. The excitation of
1 and 2 was applied at 330 nm and 321 nm, respectively, at
25 °C. The pH of the solution was measured and the fluores-
cence spectrum was obtained after each addition of small
amounts of KOH 0.1 mol dm−3 until pH 10–12.
The binding constants were calculated through the fitting of the
least–squares regression curves using the ORIGIN 6.1 program.
Results and Discussion
Synthesis and Characterization
Compound 1 was synthesized in six steps, through a synthetic
route described in Scheme 1. Firstly, 2,6–diaminopyridine
was acetylated, according to a procedure described in the
literature [55], with acetic anhydride in THF to generate
compound 3. This compound was refluxed with 5–

J Fluoresc (2012) 22:1033–1046
1037
Scheme 1 Synthetic route
for the preparation of the
1,8–naphthyridines 1 and 2.
Reagents and conditions: (a)
acetic anhydride, THF,
OMe
OMe
triethylamine, 12 h, 45 %; (b)
Meldrum’s acid + HC(OCH3)3,
reflux, 82 %; (c) diphenyl ether,
250 °C, 15 min, 84 %; (d)
POCl3, 90–95 °C, 1.5 h, 63 %;
(e) 4–methoxyphenylboronic
acid, Pd(PPh3)4, benzene,
ethanol, Na2CO3, reflux, 54 %;
(f) H2SO4 (aq) 10 %, reflux,
5 min, 85 %
H2N
N
N
N
N
HN
O
CH3
1
2
O
O
O
O
(a)
(b)
H2N
N
NH2
HN
N
NH2
N
N
H
HN
3
O
O
4
CH3
CH3
(c)
O
HN
N
N
H
O
5
OMe
OMe
CH3
(d)
Cl
(f)
(e)
HN
H2N
HN
N
N
N
N
N
N
O
O
1
2
6
CH3
CH3
methoxymethylene Meldrum’s acid (generated in situ by
refluxing Meldrum’s acid with trimethyl orthoformate) [56]
to afford compound 4, which was cyclized by means of
thermolysis in diphenyl ether at 250 °C, to generate N–(5,8–
dihydro–5–oxo–1,8–naphthyridin–2–yl)acetamide (5). The
reaction of 5 with POCl3 led to the formation of N–(5–
chloro–1,8–naphthyridin–2–yl)acetamide (6), applying a clas-
sical procedure [58, 59]. In the next step, 1,8–naphthyridine 2
was obtained through Suzuki coupling [63], which involved
the reflux of 6, 4–methoxyphenylboronic acid, and Pd(PPh3)4
in benzene, ethanol, and Na2CO3. Finally, compound 2 was
hydrolyzed by reflux with H2SO4 (10 %) for 5 min to form the
1,8–naphthyridine 1. The identity and purity of the com-
pounds 1 and 2 were verified through their characterization
using IR, NMR, and elemental analyses.
Photophysical Properties of 1 and 2
Table 1 shows experimental data obtained from the analysis of
UV–vis and fluorescence emission spectra for compounds 1
Table 1 Photophysical properties of compounds 1 and 2
Compound
Solvent
λ
max (nm)
ε
max (dm3mol−1 cm−1
)
λ
em (nm)a
Фb
1
1
1
2
2
2
DMSO
350
340
330
327
325
321
1.74×104
8.84×103
8.82×103
2.24×104
1.17×104
7.88×103
408
408
401
426
447
470
0.37
0.31
0.19
0.03
0.07
0.06
DMSO–waterc
water, pH 8.5d
DMSO
DMSO–watere
water, pH 6.0d
a The solutions were excited using absorption λmax values. b Relative to the standard quinine sulfate in 0.1 mol dm−3 of H2SO4 (λexc0350 nm; Ф00.577).
c 7:3 (v/v), buffer tris–HCl, pH 8.5. d Buffer tris–HCl. e 7:3 (v/v), buffer tris–HCl, pH 6.0

1038
J Fluoresc (2012) 22:1033–1046
and 2 in DMSO, water and in a DMSO–water (7:3, v/v)
mixture. The solution of naphthyridine 1 absorbs in the UV
range with a maximum in the wavelength (lmax) at 330 nm in
water (HCl/tris; pH 8.5) and at 350 nm in DMSO, with a
molar absorptivity of 1.74×104 dm3 mol−1 cm−1. For com-
pound 2, the maxima in the wavelengths were at 321 nm in
water (HCl/tris; pH 6.0) and 327 nm in DMSO, with a molar
absorptivity in DMSO of 2.24×104 dm3 mol−1 cm−1.
data, the band of compound 1 is slightly changed with the
change in the polarity of the medium (Δl07 nm on compar-
ing data obtained in DMSO and in water), while compound 2
shows an important bathochromic shift of 44 nm on compar-
ing the emission spectrum obtained in DMSO with that in
water. This observation suggests that compound 2 can be used
as a solvatofluorochromic probe [65–68] in the investigation
of the polarity of pure solvents and binary mixtures.
Solutions of 1 and 2 were excited using their respective
absorption lmax values, and it was observed that these com-
pounds are fluorescent. The quantum yields (Ф) were obtained
in DMSO and water (HCl/tris at pH 8.5 for 1 and pH 6.0 for 2)
and are similar to other results found in the literature related to
1,8–naphthyridines [42, 44, 64]. Compound 1 exhibits a
higher Ф value than compound 2, in DMSO and in buffered
water, which is in agreement with the well documented fact
that 1,8–naphthyridines with electron–donating groups in their
molecular structure have higher Ф values in comparison with
those possessing weaker electron–donating groups [61]. The
pKa Values of 1 and 2 in Aqueous Solution
Figure 1(a) shows the fluorescence emission spectra for
compound 1 at several pH values. The fluorescence emis-
sion band at 401 nm predominates for pH values between
5.8 and 12.0 and this band disappears at pH values below
4.0, simultaneously with the appearance of another band
with low intensity in the region of 500 nm. Figure 1(b)
shows the titration curve, as a plot of the fluorescence
emission intensity (IF) at 401 nm as a function of pH.
Figure 2(a) shows the influence of pH on the fluores-
cence emission spectrum of 2, where it can be observed that
for pH values between 4.8 and 10.0 a maximum fluores-
cence occurs at 470 nm and for low pH values, more
precisely in the pH range of 2.5–3.3, a fluorescence quench-
ing occurs without the bathochromic shift verified in the
case of compound 1. Figure 2(b) shows the corresponding
titration curve obtained from the emission spectra.
The titration curves were used to obtain the pKa values by
means of a theoretical fitting of the experimental data using
a sigmoidal equation. The pKa value for the conjugated acid
of 1 was 6.18 0.04 (S.D.02.73×10−4; r200.999) and for 2
this value was 3.83 0.12 (S.D.09.60×10−4; r200.997). A
pKa value of 3.36 was obtained by Perrin for the conjugated
acid of non–substituted 1,8–naphthyridine in water [69].
l
max values for the absorption of 1 change if the polarity of the
medium is altered, a hypsochromic shift of 20 nm being
observed on comparing the spectrum obtained in DMSO with
that in water. This is due to the fact that water is able to act as a
hydrogen–bond donating solvent, interacting strongly with the
lone electron pair of the nitrogen atom in the 2–position of 1.
This hinders the electronic transition involving the amino
group, leading to the negative solvatochromism observed.
This observation is corroborated by the fact that 2 is less
sensitive to the medium polarity, because this compound has
an acetyl bound to the amino group in its molecular structure,
which diminishes the electronic availability of the nitrogen
atom. This explanation also aids an understanding of the
reduction in the Ф values of compound 1 verified in the
presence of water. Interestingly, according to the emission
a
b
250
250
pH 2.2
pH 3.0
pH 3.8
pH 5.8
200
200
150
100
50
pH 7.0
pH 7.9
pH 8.6
pH 10.0
pH 11.0
pH 12.0
150
100
50
0
0
350
2
4
6
8
10
12
400
450
500
550
600
pH
Wavelength/nm
Fig. 1 (a) Fluorescence emission spectra of 1 at different pH values and (b) IF values for 1 at 401 nm as a function of pH

J Fluoresc (2012) 22:1033–1046
1039
a
b
350
350
300
250
200
150
100
50
2.5
2.8
3.0
3.3
4.8
6.0
7.0
8.0
9.4
10.0
300
250
200
150
100
50
0
2
0
4
6
8
10
12
400
450
500
550
600
pH
Wavelength/nm
Fig. 2 (a) Fluorescence emission spectra of 2 at different pH values and (b) IF values for 2 at 470 nm as a function of pH
Compound 1 has an amino electron–donor group in its molec-
ular structure, which is responsible for increasing the electronic
density at the nitrogen atoms of the naphthyrine ring. This may
be the reason for the higher pKa value for 1 in comparison with
2 and with the non–substituted naphthyridine. The intermediate
value obtained for 2 in comparison with the other two naph-
thyridines can be explained considering the acetamide substit-
uent as a moderate electron–releasing group.
naphthyridine is quenched, until the complete disappearance
of the band with a maximum at 401 nm. The IF values at
401 nm were collected from the spectra, as shown in Fig. 3(b),
and indicate that saturation occurs at a Cu2+ concentration of
4.0×10−3 mol dm−3. Similar results were obtained for the
titration of compound 2 with Cu2+, as shown in Fig. 4.
The titration curves showed behavior typical of a 1:1
naphthyridine:Cu2+ stoichiometry. The binding constants
were calculated by fitting the experimental data with a
nonlinear regression to eq 1, which considers the participa-
tion of one molecule of the naphthyridine to one Cu2+ ion
[70, 71].
Influence of Cu2+ on the Fluorescence of 1 and 2
Figure 3(a) shows a group of fluorescence emission spectra
relating to the titration of 1 with Cu2+. It can be observed that
with the addition of Cu2+ the fluorescence emission of the
Â
À
ÁÃ Â
ÁÃ
IF ¼ IFo þ IF11K11c Cu2þ = 1 þ K11cðCu2þ
ð1Þ
a
b
450
450
400
350
300
250
200
150
100
50
400
350
300
250
200
150
100
50
0
360
380
400
420
440
460
480
500
520
540
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
c (Cu2+)/10-3 mol dm-3
Wavelength/nm
Fig. 3 (a) Influence of the addition of increasing amounts of Cu2+ on
the fluorescence emission spectrum of 1 (c01×10−6 mol dm−3) in
DMSO–water 7:3 (v/v) at 25 °C. The final concentration of Cu2+ was
4×10−3 mol dm−3. (b) Variation in the IF of 1 at 408 nm with the
addition of increasing amounts of Cu2+

1040
J Fluoresc (2012) 22:1033–1046
a
b
400
400
350
300
250
200
150
100
50
350
300
250
200
150
100
50
0
380
400
420
440
460
480
500
520
540
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Wavelength/nm
c (Cu2+)/10-3 mol dm-3
Fig. 4 (a) Influence of the addition of increasing amounts of Cu2+ on
the fluorescence emission spectrum of 2 (c01×10−6 mol dm−3) in
DMSO–water 7:3 (v/v) at 25 °C. The final concentration of Cu2+ was
3×10−3 mol dm−3. (b) Variation in the IF of 2 at 440 nm with the
addition of increasing amounts of Cu2+
where IF is the fluorescence emission intensity after each
addition of Cu2+, c (Cu2+) is the concentration of the metal
after each addition of Cu2+, IF11 is the minimum value of IF
obtained by the addition of Cu2+, and K11 is the binding
constant. The K11 value was obtained through this mathe-
matical treatment and for compound 1 it was found to be
(1.54 0.43)×103 dm3 mol−1 (r200.998 and S.D.01.7×
10−4), while for 2 it was found to be (6.25 0.35)×
102 dm3 mol−1, (r200.996 and S.D.04.1×10−4). The K11
value for 1:Cu2+ is larger than that for 2:Cu2+ due to the
electron–releasing effect of the amino group, which
increases the electron density at the naphthyridine ring,
making the electron pairs on the nitrogen atoms of the 1–
and 8– positions of the naphthyridin ring 1 more available to
interact with the metal ion. Compound 2 has in its molecular
structure an acetamide group in the 2– position, and the
carbonyl is responsible for reducing the electron density of
the amino group through the resonance effect, leading con-
sequently to the reduction in the complexation ability of the
compound. The results obtained, in combination with data
a
b
Compound 1
1.0
1:Cu2+
CN-
Cl-
600
500
400
300
200
100
0
0.8
0.6
0.4
0.2
0.0
Br-
I-
H2PO4-
HSO4-
NO3-
F-
400
450
500
-
-
CN- Cl-
Br-
I- H2PO4- HSO4 NO3 F-
Wavelength/nm
Fig. 5 (a) Fluorescence emission spectra at 25 °C of solutions of 1:
Cu2+ in DMSO–water (7:3; v/v) in the presence of various anions. (b)
IF for the anions added to solutions of 1 at 408 nm relative to the CN−
added. A λexc value of 340 nm was used and the concentrations of the
species were: c (1)01×10−6 mol dm−3, c (Cu2+)04×10−3 mol dm−3
and c (anion)08×10−3 mol dm−3
,

J Fluoresc (2012) 22:1033–1046
1041
a
b
1.0
0.8
0.6
0.4
0.2
0.0
Compound 1
1:Cu+
CN-
Cl-
700
600
500
400
300
200
100
Br-
I-
H2PO4-
HSO4-
NO3-
F-
0
400
450
Wavelength/nm
500
550
-
-
CN- Cl-
Br-
I- H2PO4- HSO4 NO3 F-
Fig. 6 (a) Fluorescence emission spectra at 25 °C of solutions of 1:
Cu+ in DMSO–water (7:3; v/v) in the presence of various anions. (b) IF
for the anions added to solutions of 1 at 408 nm relative to the CN−
added. A λexc value of 340 nm was used and the concentrations of the
species were: c (1)01×10−6 mol dm−3, c (Cu+)03×10−3 mol dm−3
and c (anion)03×10−3 mol dm−3
,
in the literature for the complexation of 1,8–naphthyridines
with metal ions [42, 44, 72–74], suggest that the coordina-
tion of Cu2+ with compound 1 occurs in the naphthyridine
center, at the nitrogens in the 1– and 8– positions. The
coordination causes the quenching in the fluorescence of
the system through a photoinduced electronic transfer
(PET), which can occur between Cu2+ and the fluorophore.
This situation is reported in the literature as involving the
complexation of fluorescent compounds with metal ions
[75–77].
when Cu2+ was used. The experimental data were fitted with
eq. (1), leading to K110(2.50 0.11)×103 dm3 mol−1 (r200.996
and S.D.03.4×10−4) for 1 while the K11 value for 2 was (7.25
0.23)×102 dm3 mol−1 (r200.999 and S.D.04.1×10−5).
Competition Assay Using the Complex
Between the Naphthyridines and Cu2+ as an Anionic
Fluorogenic Chemosensor
According to the literature, Cu2+ reacts with CN− to form
CuCN and cyanogen (CN)2 [78]. Subsequently, complexes
with a high stability constant of the type [Cu(CN)2]−, [Cu
Compounds 1 and 2 were also titrated with Cu+ and the
pattern observed in these assays was very similar to that verified
a
b
700
700
600
500
400
300
200
100
0
600
2 equiv. CN-
500
1 equiv. CN-
400
300
200
100
0
1
2
3
4
5
6
7
8
9
10
380
400
420
440
460
480
500
520
540
c (CN-)/10-3 mol dm-3
Wavelength / nm
Fig. 7 (a) Influence of the addition of increasing amounts of CN− on the
fluorescence emission spectrum of 1:Cu2+ in DMSO–water 7:3 (v/v) at
25 °C. The final concentration of CN− was 8×10−3 mol dm−3. (b)
Variation in the IF of 1:Cu2+ at 408 nm with the addition of increasing
amounts of CN−. A λexc value of 340 nm was used and the concentrations
of 1 and Cu2+ were 1×10−6 mol dm−3 and 4×10−3 mol dm−3, respectively

1042
J Fluoresc (2012) 22:1033–1046
a
b
800
700
600
500
400
300
200
100
800
700
600
500
400
300
200
100
0
1 equiv. CN-
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
380
400
420
440
460
480
500
520
c (CN-)/10-3 mol dm-3
Wavelength/nm
Fig. 8 (a) Influence of the addition of increasing amounts of CN− on
the fluorescence emission spectrum of 1:Cu+ in DMSO–water 7:3 (v/v)
at 25 °C. The final concentration of CN− was 3×10−3 mol dm−3. (b)
Variation in the IF of 1: Cu+ at 408 nm with the addition of increasing
amounts of CN−. A λexc value of 340 nm was used and the concen-
trations of 1 and Cu+ were 1×10−6 mol dm−3 and 3×10−3 mol dm−3
respectively
,
(CN)3]2–, and [Cu(CN)4]3–are formed in excess of CN−, and
the species commonly found are [Cu(CN)2]− (K01×1024)
and [Cu(CN)4]3– (K01×1030) [79–83]. This finding is the
basis for the recent development of some anionic chemo-
sensors based on displacement assays [24, 84–87]. There-
fore, the potential of the complexes comprised of 1,8–
naphthyridines and copper ion as anionic chemosensors
was studied in a DMSO–water mixture. The strategy used
here is based on the idea that an anion having a great affinity
for the metal ion can displace the latter from the naphthyr-
idine site, leading to the restoration of the fluorescence
emission in solution.
Figure 5(a) shows the influence of various anions, at the
same concentration, on the fluorescence emission spectrum
of 1:Cu2+. It can be observed that of the anions used, only
CN− was able to fully restore the original fluorescence of 1,
confirming the initial expectation. Figure 5(b) shows the
relative emission intensities for the same system in the pres-
ence of the anions, which verifies that CN− recovers 100 % of
the original fluorescence of 1, followed by H2PO4− (50 %) and
F− (32 %), while the other anions have practically no influence
on the spectrum of the complex. Very similar results were
obtained for compound 2. In addition, the fluorescence recov-
ery process is very fast: only one addition of CN− to the
complex of 1 (or 2) with Cu2+ in a concentration twice the c
(Cu2+) immediately causes the return to the original fluores-
cence of the naphthyridine in solution.
Figure 6(a) illustrates the behavior of 1:Cu+ in the presence
of several anions. It can be observed at this time that the
system becomes more selective toward CN− in comparison
with the other anions: the fluorescence emission is completely
restored when CN− is used while the influence of H2PO4− and
F− in terms of a return of the fluorescence is small (25 % and
16 % for H2PO4− and F−, respectively). One reason for the high
selectivity obtained for CN− is related to the fact that the free
−
energies of hydration for H2PO4 (−465 kJ mol−1) and
F− (−465 kJ mol−1) are high in comparison to that observed for
CN− (−295 kJ mol−1) [88, 89], and some papers have reported
the use of this strategy to make chemosensors selective to CN− in
solution [90–95]. Thus, H2PO4− and F− in aqueous medium are
preferentially solvated by water, hindering their interaction with
Cu2+. Another explanation that can be offered makes use of the
hard and soft acids and bases (HSAB) theory of Pearson [96, 97].
Cu+ is a soft acid which forms a complex with CN− of high
Scheme 2 Representation
OMe
OMe
OMe
for the displacement assay
studied with the addition
of increasing amounts of
CN– to the complex 1:Cu2+
2 CN-
H2N
Cu(II)
[Cu(CN)2]-
+
H2N
N
N
H2N
N
N
N
N
Cu(II)
non fluorescent
fluorescent
fluorescent

J Fluoresc (2012) 22:1033–1046
1043
stability. H2PO4− and F− are hard bases, and Cu2+ is a harder acid
than Cu+, which can aid an explanation of the differences be-
tween the results obtained in the two systems studied. Hence, the
experimental data suggest two different mechanisms of interac-
addition of CN−, 1 is released from the metal and becomes free
to exhibit its fluorescence.
−
tion for the anionic species with the fluorogenic system. H2PO4
Conclusions
and F− would displace the metallic ion from the naphthyridinic
site, while CN− would react with Cu2+ changing its oxidation
state before the displacement of the metal.
1,8–Naphythyridines 1 and 2 were synthesized and they
were found to be fluorescent in solution. In the presence
of Cu2+ (and Cu+) their fluorescence was quenched, due to a
PET mechanism between the metallic ion and the fluoro-
phore. The assays employing the complex of the naphthyr-
idines with the copper ions in the presence of various anions
showed both selectivity and the complete and immediate
return of the original fluorescence with the addition of CN−.
In addition, the titration experiment demonstrated that two
anion equivalents are needed for the complete restoration of
the fluorescence of the naphthyridines. The first equivalent
of the anion is needed to change the oxidation state of the
metal to Cu+, and the second equivalent is responsible for
displacing the metallic ion from the binding site in the naph-
thyridine, making the latter free to fluoresce. This was con-
firmed with the titration of 1 using Cu+, which revealed that
only one equivalent of the anion is needed to completely
restore the fluorescence of 1.
Figure 7(a) shows a set of fluorescence emission
spectra related to the titration of 1:Cu2+ with CN−,
where it can be verified that with the addition of the
anion there occurs an increase in the IF corresponding
to the appearance of the free naphthyridine at 408 nm.
The experimental data were used to obtain a plot of the
IF values at 408 nm as a function of c (CN−), shown in
Fig. 7(b). The titration curve obtained has a sigmoidal
profile, which suggests that the addition of the anion
has initially a small influence on the increase in the
fluorescence and on the addition of larger amounts of
the anion the fluorescence is restored, corresponding to
the same value obtained for 1 prior to the addition of
the metal. A careful analysis of the titration curve
reveals that two equivalents of the anion need to be
added to fully restore the fluorescence of the system.
When two straight lines are placed tangentially to the
two halves of the curve, it is verified that their inter-
section coincides with one equivalent of anion added.
The data suggest that the first anion equivalent is used
to change the oxidation state of the metallic ion, from
Cu2+ to Cu+, and in sequence the second anion equiv-
alent is responsible for displacing the metal from the
naphthyridine binding site, which leads to the full return
of the fluorescence of the system. A very similar result
was obtained for the system with compound 2, which
can be explained by the fact that both the reduction of
Cu2+ and its displacement by the anion are very fast–
occurring events.
The strategy applied for the detection of CN− in aqueous
medium is easy to perform, but the range of detection of CN−
is in the order of 10−4 mol dm−3, which is above the minimum
concentration of CN− allowed by the World Health Organiza-
tion in potable water, that is 1.7×10−6 mol dm−3 [98]. Never-
theless, the potential of the assay studied is demonstrated
herein, as well as the possibility to exploit a clever design to
synthesize other modified 1,8–naphthyridines in order to
change certain features, such as to increase the quantum yield
values and the binding constants associated with the complex-
ation of these compounds with metallic ions, which will
improve the sensitivity of this methodology.
Figure 8(a) shows the fluorescence emission spectrum for the
titration of 1:Cu+ with CN−, which reveal an increase in the
fluorescence of the system on the addition the anion,
corresponding to the appearance of the free naphthyridine. The
corresponding titrating curve, shown in the Fig. 8(b), presents a
sigmoidal shape, similar to that obtained for the titration of the
Cu2+ complex. However, the data show that only one CN−
equivalent is needed to displace Cu+ from the naphthyridine
site, causing a complete return to the original fluorescence. Thus,
these data reinforce the above suggestion regarding the role of
CN− in the change in the oxidation state of Cu2+.
Acknowledgments The financial support of Brazilian Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
FAPESC, INCT–Catálise, UFSC, and FURB is gratefully acknowledged.
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