Microwave-assisted synthesis of 3-hydroxy-4-pyridinone/naphthalene conjugates. Structural characterization and selection of a fluorescent ion sensor

Article abstract of DOI:10.1016/j.tet.2010.08.065

Two novel 3-hydroxy-4-pyridinone/naphthalene conjugates (L1 and L2) with different distances between the chelating and the fluorescent moieties were synthesized using conventional heating and microwave irradiation achieving a shorter reaction time. The structure of both compounds was confirmed by X-ray crystallography, revealing that these compounds were isolated as hydrochloride salts in dihydroxypyridinium forms. In solution and in the presence of a base, the tautomeric keto forms may be obtained as it was elucidated by NMR analysis. The dihydroxypyridinium form of L1 exhibits fluorescence at 450 nm, both in ACN and DMSO, whereas the corresponding keto form exhibits fluorescence at 365 nm. In contrast, the dihydroxypyridinium form of L2 only fluoresces in DMSO, exhibiting a band at 340 nm, while the keto form is non-fluorescent. These distinct fluorescent behaviors reveal that the tautomeric form in which the ligands are isolated and the distance between the chelating and fluorescent functions strongly influences their fluorescence properties. Ligand L1 exhibits better fluorescence properties and its fluorescence intensity is quenched in the presence of variable concentration of Cu²⁺, Zn²⁺, and Fe³⁺, thus making it suitable to be used as ion sensor.

Full text of DOI:10.1016/j.tet.2010.08.065

Tetrahedron 66 (2010) 8544e8550
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Microwave-assisted synthesis of 3-hydroxy-4-pyridinone/naphthalene
conjugates. Structural characterization and selection of a fluorescent ion sensor
,
a
a
a
c
c
Ana M.G. Silva a , Andreia Leite , Mariana Andrade , Paula Gameiro , Paula Brandão , Vítor Felix ,
*
Baltazar de Castro a, Maria Rangel b
,
*
a Requimte, Departamento de Química, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal
b Requimte, Instituto de Ciências Biomédicas Abel Salazar, 4099-003 Porto, Portugal
c Department of Chemistry, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2010
Received in revised form 24 August 2010
Accepted 28 August 2010
Available online 24 September 2010
Two novel 3-hydroxy-4-pyridinone/naphthalene conjugates (L1 and L2) with different distances be-
tween the chelating and the fluorescent moieties were synthesized using conventional heating and
microwave irradiation achieving a shorter reaction time. The structure of both compounds was con-
firmed by X-ray crystallography, revealing that these compounds were isolated as hydrochloride salts in
dihydroxypyridinium forms. In solution and in the presence of a base, the tautomeric keto forms may be
obtained as it was elucidated by NMR analysis. The dihydroxypyridinium form of L1 exhibits fluorescence
at 450 nm, both in ACN and DMSO, whereas the corresponding keto form exhibits fluorescence at
365 nm. In contrast, the dihydroxypyridinium form of L2 only fluoresces in DMSO, exhibiting a band at
340 nm, while the keto form is non-fluorescent. These distinct fluorescent behaviors reveal that the
tautomeric form in which the ligands are isolated and the distance between the chelating and fluorescent
functions strongly influences their fluorescence properties. Ligand L1 exhibits better fluorescence
Keywords:
3-Hydroxy-4-pyridinones
Dihydroxypyridinium forms
Naphthalene dyes
Fluorescent sensors
properties and its fluorescence intensity is quenched in the presence of variable concentration of Cu2þ
,
Zn2þ, and Fe3þ, thus making it suitable to be used as ion sensor.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
transition metals, like Zn(II), Cu(II), and VO(II). In this context, Zn(II)
and VO(IV) 3,4-HPO complexes are currently under study as orally
active insulin enhancing drugs.4,7 The pyridinone structure allows
the synthesis of ligands with variable lipophilicity making them
useful not only for pharmaceutical applications but also as extrac-
tants of metal ions from organic matrices.4 Fluorescence tech-
niques, particularly those making use of 3,4-HPO fluorescent
probes have become increasingly important for the detection of
intracellular metal ions in several diseases related with metal ion
burdens.8
Taking into account the versatile chemistry and the potential
clinical application of (3,4-HPO)s, two novel bidentate ligands were
prepared both bearing a 3,4-HPO chelator moiety covalently bound
to a mono-substituted naphthalene fluorescent platform. The
choice of the naphthalene platform is related to its low molecular
weight and biological compatibility. Indeed, many naphthalene-
based fluorophores have been used to prepare fluorescent sensors9
and other fluorescent frameworks.10 Also, the naphthalene skeleton
is present in a large number of clinical drugs, such as naphazoline,
which is a potent cardiovascular agent, and naproxen, an anti-in-
flammatory agent.11 Recently, other important pharmacological
properties, such as potential antitumor and antiprotozoal activities
were reported for other naphthalene systems.12
Analytical assays based on functionalized fluorophores are
ubiquitous in biotechnology, medicinal chemistry, and environ-
mental sciences.1,2 In these fields, the design of functionalized
molecules, with variable lipophilicity, whose fluorescence pro-
perties change in the presence of target metal ions allows to study
aspects of metal ion accumulation, trafficking and function or
toxicity in living systems in real-time.3 With this motivation,
considerable efforts have been made, in the past few years, to
synthesize new simple and easy-to-make sensors able to respond
with high sensitivity and efficiency to the presence of different
metal ions.
3-Hydroxy-4-pyridinones (3,4-HPO)s constitute
a class of
ligands with clinical application due to their high affinity towards
several biological important metal ions.4 These compounds have
been widely used as chelating agents for the treatment of diseases
related with iron overload, such as b
-talassemia,5 and other clinical
situations related to the central nervous system injuries caused by
Al(III) surplus.6 (3,4-HPO)s also possess a high affinity for divalent
* Corresponding authors. E-mail address: ana.silva@fc.up.pt (A.M.G. Silva).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.08.065

A.M.G. Silva et al. / Tetrahedron 66 (2010) 8544e8550
8545
In order to understand the effect of proximity between the
chelating and fluorescent functions in the fluorescence properties
of naphthalene, we designed ligand L1 (Scheme 1), which contains
the chelating unit and the fluorophore directly attached through
the nitrogen atom of the pyridinone ring, and ligand L2 (Scheme 2)
in which the same functions are separated by a eCH2CH2CONHe
bridge, thus generating a more flexible structure.
reaction was conducted under microwave irradiation (60 min,
55 ꢀC), conjugate 4 was obtained in a significant shorter period of
time with similar yield. Subsequent hydrogenolysis, under hydro-
gen atmosphere in the presence of Pd/C (10%) and HCl, produced
the hydrochloride salt of L2, with a 65% yield.
In principle, the (3,4-HPO)s are able to exist in two tautomeric
forms (enol and keto forms).14 In the present work, both ligands L1
and L2 were isolated as hydrochloride salts in dihydroxypyridinium
forms and for both compounds crystal with diffractometric quality
were obtained.
NH2
O
O
N
OBn
OBn
HCl/ MeOH
+
Δ or MW
O
2.2. X-ray diffraction
1
OH
Confirmation of the dihydroxypyridinium forms was obtained
from X-ray diffraction data obtained from crystals of both ligands
L1 and L2 (Figs. 1 and 2). The crystal structure of L1 is built up from
an asymmetric unit composed of one L1 cation and one chloride
anion. Molecules of L1 are linked in to 1-D polymeric structure
through OeH/Cl hydrogen bonding interactions with O/O dis-
tances of 2.914(2) and 3.040(2) Å with OeH/Cl angles of 175(4)
and 154(3)ꢀ, respectively (see Fig. 1, bottom view). Also, the
asymmetric unit of L2 is composed of one molecule of L2 and one
chloride counter ion, which are assembled into a 1-D network of
hydrogen bonding interactions as shown in Fig. 2. The chloride
anion is bonded to two phenol groups and one NeH amide group of
neighboring L2 cations with Cl/O distances of 2.988(2) and 3.015
(2) Å. The corresponding OeH/O angles are 174(4) and 172(5)ꢀ,
respectively and NeH/O angle is 177(3) Å.
4
OH
3
5
6
2
BCl3, CH2Cl2
N
Cl-
2'
8'
5'
7'
6'
3'
4'
L1
Scheme 1. Synthesis of hydrochloride salt of ligand L1.
O
O
NH2
OBn
OBn
DCC, HOBt
rt or MW
+
N
N
HO
O
NH
O
3
4
OH
4
6' 7'
OH
3
5
H
2, Pd/ C
5'
4'
8'
6
EtOH, HCl
Cl-
L2
N
NH
3' 2'
O
Scheme 2. Synthesis of hydrochloride salt of ligand L2.
2. Results and discussion
2.1. Synthesis
Fig. 1. X-ray single crystal structure of L1$HCl showing two different features: top, the
molecular of L1; bottom, the assembly of L1 molecules by OeH/Cl hydrogen bonding
interactions into 1-D polymeric structure.
Ligand L1 was efficiently synthesized using a two sequence
steps protocol (Scheme 1). First, 1-naphthylamine was allowed to
react with 3-benzyloxy-2-methyl-4-pyrone 1, in a HCl/methanol
mixture under reflux (oil-bath) for 24 h, giving rise to derivative 2
in 52% yield. In recent literature reports, it has been demonstrated
that many synthetic protocols, in particular protocols regarding the
synthesis of organic dyes, can be significantly improved by micro-
wave heating.13 Taking into account the potential advantages of the
direct and rapid microwave heating process, the condensation re-
action was conducted using microwave heating under closed vessel
conditions. Indeed, increasing the reaction temperature to 140 ꢀC,
the reaction leads to derivative 2 in only 60 min, providing a sig-
nificant reduction of reaction time. Debenzylation was performed
with BCl3 in dichloromethane, under an argon atmosphere,
affording the expected hydrochloride salt of ligand L1 in 82% yield.
In order to prepare ligand L2 (Scheme 2), the coupling reaction
of 1-naphthylamine with 3-benzyloxy-1-(30-carboxypropyl)-2-
methyl-4-pyridinone 3 was carried out through in situ generation
of the corresponding activated ester using N,N0-dicyclohex-
ylcarbodiimide (DCC) and hydroxybenzotriazole (HOBt). After
stirring at room temperature for 24 h, the reaction provided
naphthaleneepyridinone conjugate 4 in 31% yield. When the same
Fig. 2. X-ray single crystal structure of L2.HCl showing two different features: left the
molecular of L2; right, the network of OeH/Cl and OeH/Cl hydrogen bonding
interactions.
The spatial disposition of the dihydroxypyridine and naph-
thalene entities is different in L1 and L2 cations. For L1, the two
aromatic fragments are twisted giving a dihedral angle between
their planes of 77.98(5)ꢀ, which prevents electronic communica-
tion between them in solid state. For L2, the dihydroxypyridine
intercepts the naphthalene entity at a dihedral angle of 39.43(5)ꢀ

8546
A.M.G. Silva et al. / Tetrahedron 66 (2010) 8544e8550
and CeH from naphthalene points to dihydroxypyridine at a dis-
tance of 2.974(3) Å, which seems to suggest the existence of an
appear at 145.6 and 170.4 ppm (Fig. 3d). These chemical shift values
are in agreement with the chemical shifts observed for the parent
3-benzyloxy-4-pyridinone derivative 2, where protons H-5 and H-6
appear at 6.31 and 7.62 ppm and carbons C-3 (CeOBn) and C-4 (C]
O) appear at 144.4 and 172.5 ppm.
edge to face CeH/p interaction. The average CeO quinone bond
distance of 1.254(3) Å is typical of a carbonyl group while the CeO
distances of 1.333(3) and 1.352(3) Å in L1 and 1.342(3) and 1.350
(3) Å in L2 are clearly single bond distances consistent with the
presence of phenol groups, as required by the charge balance of
the molecular formulas of both organic salts. X-ray single crystal
structure of derivative 4 was also obtained and is supplied in
Supplementary data.
Similar results were observed for ligand L2 (see Experimental
section).
Although these ligands are sparingly soluble in water, once
dissolved in DMSO it is possible to prepare aqueous solutions (99%
H2O/1%DMSO) and determine the dissociation constants of L1 and
L2 by spectrophotometric titration. Isolated as hydrochloride salts,
the ligands possess two dissociable protons, which correspond to
two hydroxyl groups (Scheme 3).
2.3. Physico-chemical properties in solution
The structure of ligands L1 and L2 observed in solution was
established by NMR analysis (1H and 13C, 1D, and 2D experiments,
including COSY, HSQC, and HMBC spectra for unequivocal assign-
ment of proton and carbon chemical shifts). The 1H NMR spectrum
of L1 in DMSO-d6 (Fig. 3a) exhibits two doublets at 7.55 ppm and
8.32 ppm (3J5,6-H¼6.8 Hz), corresponding to H-5 and H-6. While H-
5 shows strong HMBC correlation with a signal at 143.2 ppm
assigned as C-3 (CeOH), H-6 shows HMBC correlation with a signal
at 161.0 ppm assigned to C-4 (CeOH) (Fig. 3c). These results provide
evidence of the presence of the dihydroxypyridinium form. In order
to investigate the presence of other tautomeric forms in solution,
solvents having different polarities such as acetonitrile (aprotic
solvent) and methanol (protic solvent) were employed.15 Indeed,
both in acetonitrile-d3 (exhibiting H-5 at 7.38 ppm, H-6 at
7.96 ppm; C-3 at 145.4 ppm and C-4 at 161.8 ppm) and in methanol-
O
O
OH
R1
O
N
R1
N
R2
R2
OH
OH
R1
pK
pK
a2
a1
N
R2
H
O
O
O
O
R1
N
R1
N
R2
R2
H2L+
L-
HL
d4 (exhibiting H-5 at d 7.36 ppm, H-6 at 8.25 ppm; C-3 at 145.1 ppm
Scheme 3. Dissociation steps of ligands.
and C-4 at 161.5 ppm), the dihydroxypyridinium form is the only
detectable form in solution. The tautomeric keto form was only
achieved when the pH was increased by adding K2CO3 directly in
the NMR tube. This fact is clearly seen by analysis of the resonance
positions of protons H-5 and H-6, which at higher pH appear at
lower frequencies, 6.31 and 7.63 ppm (Fig. 3b), and by the reso-
nance positions of carbons C-3 (CeOH) and C-4 (C]O), which
The values obtained for these two acidity constants are
pKa1¼3.01ꢁ0.05 and pKa2¼9.61ꢁ0.01 for L1, and pKa1¼3.27ꢁ0.03
and pKa2¼9.86ꢁ0.9 for L2 and the distribution diagrams as a func-
tion of pH are provided in the Supplementary data. The values are
within the usual ranges observed for (3,4-HPO)s centered on 3 and
9.4. The slightly lower pKa values obtained for L1, when compared
to those obtained for L2, are probably due to the close proximity of
the aromatic naphthalene skeleton to the pyridinone heterocyclic
ring, which provides an electron-withdrawing effect and a con-
comitant decrease of the protonation constant.
The UV/visible spectra of both ligands (Fig. 4) show an intense
band at ca. 290 nm, which is characteristic of the pyridinone
system. A shoulder at ca. 325 nm, assigned to the naphthylamine
p
p
system is discernible for L1 but not for L2 thus indicating the in-
fluence of the two spacers as observed in the X-ray data.
1.0
0.8
0.6
0.4
0.2
0.0
275
300
325
350
375
400
λ(nm)
Fig. 3. Aromatic region of NMR spectra of ligand L1: (a) 1H NMR in DMSO-d6, (b)
1H NMR in DMSO-d6þK2CO3, (c) 13C NMR in DMSO-d6, and (d) 13C NMR in DMSO-
d6þK2CO3.
Fig. 4. UV/visible spectra of ligands L1 and L2 together with those of naphthylamine
and a 3,4-HPO in DMSO.

A.M.G. Silva et al. / Tetrahedron 66 (2010) 8544e8550
8547
800
As observed for most fluorophores the fluorescence properties
of naphthylamine are solvent dependent as illustrated in Fig. 5. The
naphthylamine fluorescence is observed at 450 nm in water,
425 nm both in methanol and ethanol, 413 nm in ACN and 427 nm
in DMSO, showing that this fluorophore is fluorescent in both protic
and aprotic solvents.
L1 Keto
L1 Enol
600
400
200
0
900
800
ACN
700
600
500
400
300
200
100
0
DMSO
EtOH
H2O
MeOH
300
350
400
450
500
550
(nm)
Fig. 7. Fluorescence spectra of enol and keto forms of L1 in DMSO.
Once recognized the more interesting fluorescence properties
of ligand L1, the fluorescence behavior of this ligand was studied
in the presence of the metal ions Cu2þ, Zn2þ, and Fe3þ in order to
evaluate its potential as ion sensor. Fluorescence intensity of L1
was quenched upon titration of Cu2þ, Zn2þ, and Fe3þ (see Fig. 8
for fluorescence quenching of L1 in the presence of Cu2þ and
350
400
450
500
550
600
(nm)
Fig. 5. Fluorescence spectra of naphthylamine in different solvents.
Fig. 9 for fluorescence quenching of L1 in the presence of Cu2þ
,
The fluorescence properties of the new ligands based on the
naphthylamine fluorophore are also solvent dependent and sig-
nificantly altered by the bound pyridinone. Ligand L1 is fluorescent
Zn2þ, and Fe3þ).
300
200
100
0
(
lem¼450 nm) both in ACN and DMSO but it is non-fluorescent in
water, methanol and ethanol, revealing that L1 is only fluorescent
in aprotic solvents. In contrast, ligand L2 only fluoresces in DMSO
exhibiting an emission band at 340 nm. This distinct fluorescence
behavior shows that the fluorescence properties are strongly de-
pendent on the distance between the naphthalene and the pyr-
idinone skeletons. Fluorescence spectra obtained for solutions of L1
prepared by dissolving the dihydroxypyridinium form of the ligand
in ACN are depicted in Fig. 6. The corresponding fluorescence
spectra in DMSO are provided in the Supplementary data.
350
400
450
500
550
(nm)
Fig. 8. Fluorescence quenching of L1 in ACN, in the presence of Cu2þ
.
Fig. 6. Fluorescence spectra of dihydroxypyridinium form of L1 in ACN.
The synthesized ligands show interesting fluorescence proper-
ties which depend on the keto and enol tautomeric forms. Con-
sidering that the two forms have different fluorescence behavior,
the keto forms of both ligands L1 and L2 were isolated as described
in Experimental section. In fact, the keto form of L1 is fluorescent at
365 nm while the keto form of L2 is non-fluorescent. Fluorescence
spectra obtained for solutions of enol and keto forms of L1 in DMSO
are depicted in Fig. 7.
Fig. 9. Fluorescence quenching of L1 in ACN, in the presence of Cu2þ, Zn2þ, and Fe3þ
.

8548
A.M.G. Silva et al. / Tetrahedron 66 (2010) 8544e8550
3. Conclusions
in the cavity of a CEM microwave reactor. The reaction was irra-
diated at 140 ꢀC (1 min ramp to 140 ꢀC and 60 min hold at 140 ꢀC,
using 150 W maximum power). Using a similar work-up to that
described above, compound 2 was obtained in 19.6 mg (50% of
yield). [Found: C, 79.21; N, 4.35; H, 5.58. C23H19NO2$1/2H2O re-
quires C, 78.84; N, 4.00; H, 5.75%]; dH (400 MHz; DMSO-d6) 1.58
(3H, s, CH3), 5.10 (1H, d, J 11.0 Hz, CH2C6H5), 5.27 (1H, d, J 11.0 Hz,
CH2C6H5), 6.31 (1H, d, J 7.6 Hz, 5-H), 7.01 (1H, dd, J 8.4 and J 1.0 Hz,
20-H), 7.32e7.43 (5H, m, CH2C6H5), 7.58e7.68 (4H, m, naph-H),
7.62 (1H, d, J 7.6 Hz, 6-H), 8.10 and 8.13 (2H, 2dd, J 7.4 and J 1.7 Hz,
naph-H). dC (100 MHz; DMSO-d6) 13.1 (CH3), 71.6 (CH2), 116.1 (C-
5), 121.1 (C-20), 125.5, 125.7, 127.1, 127.9, 128.1, 128.2, 128.5, 128.8,
129.3, 129.8, 133.6, 137.3, 137.4, 140.0 (C-6), 141.3 (C-2), 144.4 (C-
3), 172.5 (C-4). m/z (MS, FAB) 342 (MþH)þ.
Two 3-hydroxy-4-pyridinone/naphthalene conjugates L1 and L2
having different distances between the chelating and fluorescent
functions were conveniently synthesized using simple and easy-
to-make protocols. Both conjugates were isolated in dihydroxy-
pyridinium forms but, in solution and in the presence of a base, the
tautomeric keto forms can be also obtained. The fluorescence
properties of the ligands are strongly dependent on the tautomeric
form in which ligands are isolated and also on the distance between
the chelating and fluorescent functions. Therefore ligand L1 was
selected as the compound with more interesting fluorescence
properties. In addition, the fluorescence quenching in the presence
of variable concentration of Cu2þ, Zn2þ, and Fe3þ, demonstrates that
L1 may be used as a fluorescent ion sensor to detect and quantify
those biologically important metal ions. The results obtained in this
study are being explored in order to produce other fluorescent (3,4-
HPO)s ligands and evaluate their fluorescence behavior.
4.4. Synthesis of 3-hydroxy-2-methyl-1-naphthyl-4-
pyridinone hydrochloride (L1)
A 1 M solution of boron trichloride in dichloromethane (9.4 mL)
was dropped slowly into an ice-bath-cooled suspension of 2 (0.80 g,
2.35 mmol) in dry dichloromethane (47 mL), under an argon at-
mosphere. The mixture was stirred at room temperature for 5 h.
Methanol (15 mL) was added to quench the reaction. After removal
of the solvent in vacuum, the residue was precipitated with
methanol/acetone to afford the hydrochloride salt of L1 (0.55 g,
4. Experimental section
4.1. General information
Reagents and solvents were purchased as reagent-grade, and
used without further purification unless otherwise stated. NMR
spectra were recorded with Bruker Avance III 400 spectrometer
82%) as
a pale violet solid. Dihydroxypyridinium form mp
(400.15 MHz for 1H and 100.63 MHz for 13C). Chemical shifts (
d) are
267e270 ꢀC. [Found: C, 66.66; N, 4.90; H, 4.77. C16H13NO2$HCl re-
quires C 66.79, N 4.90, H 4.87%]; dH(400 MHz; DMSO-d6) 2.05 (3H, s,
CH3), 7.24 (1H, dd, J 7.8 and J 1.2 Hz, 20-H), 7.55 (1H, d, J 6.8 Hz, 5-H),
7.65 (1H, dt, J 7.8 and J 1.2 Hz, naph-H), 7.72 (1H, dt, J 7.8 and J 1.2 Hz,
naph-H), 7.77 (1H, t, J 7.8 Hz, naph-H), 7.86 (1H, dd, J 7.8 and J 1.2 Hz,
naph-H), 8.19 and 8.28 (2H, d, J 7.8 Hz, naph-H), 8.32 (1H, d, J 6.8 Hz,
6-H), 9.77e11.75 (2H, br, OH). dC (100 MHz; DMSO-d6) 13.8 (CH3),
111.0 (C-5), 121.0 (C-20), 125.1, 125.7, 127.6, 127.8, 128.7, 128.9, 131.0,
133.6, 136.8, 139.2 (C-6), 141.5 (C-2), 143.2 (C-3), 161.0 (C-4). m/z
(MS, FAB) 252 (MþH)þ.
reported in parts per million and coupling constants (J) in hertz;
internal standard was TMS. Unequivocal 1H assignments were made
with aid of 2D gCOSY (1H/1H), while 13C assignments were made on
the basis of 2D gHSQC (1H/13C) and gHMBC experiments (delay for
long range J C/H couplings were optimized for 7 Hz). Mass spectra
were acquired by Unidade De Espectrometria De Masas of Santiago
de Compostela and microanalyses were acquired by Unidad De
Análisis Elemental of Santiago de Compostela. Flash chromatogra-
phy was carried out using silica gel Merck (230e400 mesh). Ana-
lytical TLC was performed on precoated sheets with silica gel
(0.2 mm thick, Merck). Melting points were measured in a glass
capillary tube on a Stuart Scientific SMP1 apparatus and are
uncorrected.
The hydrochloride salt of L1 (50 mg; 0.174 mmol) was dissolved
in a (10:1) mixture of chloroform/methanol and washed twice with
a saturated aqueous solution of Na2CO3. Then the organic layer was
extracted with chloroform and dried over anhydrous Na2SO4. The
solvent was evaporated in vacuum and the resulting residue was
crystallized in chloroform/hexane to give quantitatively L1 in keto
form. Keto form mp 240e243 ꢀC. [Found: C, 74.76; N, 5.24; H, 5.01.
C16H13NO2$1/3H2O requires C, 74.69; N, 5.44; H, 5.35%] calcd dH
(400 MHz; DMSO-d6) 1.80 (3H, s, CH3), 6.31 (1H, d, J 7.2 Hz, 5-H),
7.24 (1H, dd, J 8.0 and J 1.6 Hz, 20-H), 7.63 (1H, d, J 7.2 Hz, 6-H),
7.63e7.72 (4H, m, naph-H), 8.12e8.19 (2H, m, naph-H). dC
(100 MHz; DMSO-d6) 13.0 (CH3), 111.7 (C-5), 121.8 (C-20), 126.2,
126.3, 127.7, 128.8, 129.1, 129.97, 129.98, 130.4, 134.2, 138.0 (C-2),
138.9 (C-6), 145.6 (C-3), 170.4 (C-4).
4.2. Synthesis
3-Benzyloxy-2-methyl-4-pyrone (1) and 3-benzyloxy-1-(30-
carboxypropyl)-2-methyl-4-pyridinone (3) were prepared as de-
scribed in literature.16
4.3. 3-Benzyloxy-2-methyl-1-naphthyl-4-pyridinone (2)
(a) Using oil-bath reflux: To a mixture of 3-benzyloxy-2-
methyl-4-pyrone 1 (0.51 g, 2.40 mmol), 1-naphthylamine (0.51 g,
3.57 mmol, 1.5 equiv), and methanol (2 mL) was added a 0.2 M
solution of HCl (10 mL) and the resulting solution was refluxed for
24 h. After that time, the reaction mixture was neutralized with
a solution of NaOH 5% and it was extracted twice with chloroform.
The organic layer was washed with water and then dried over
anhydrous Na2SO4. Upon evaporation of the solvent, the residue
was purified by flash chromatography using chloroform as eluent
to remove a small amount of unchanged starting material, fol-
lowed by a mixture of chloroform/methanol (95:5) to give 2.
Compound 2 was further crystallized in chloroform/hexane to
give a pale violet solid (0.42 g, 52%). (b) Using microwave irradi-
ation: A mixture of 3-benzyloxy-2-methyl-4-pyrone 1 (25.0 mg,
0.12 mmol), 1-naphthylamine (24.8, 0.17 mmol, 1.5 equiv),
methanol (0.1 mL), and a 0.2 M solution of HCl (0.5 mL) was
placed in a 10 mL reaction vial, which was then sealed and placed
4.5. Synthesis of 1-(N-naphthylcarbamoylpropyl)-3-
benzyloxy-2-methyl-4-pyridinone (4)
(a) using standard conditions: A mixture of 3-benzyloxy-1-(30-
carboxypropyl)-2-methyl-4-pyridinone
3 (1.39 g, 4.84 mmol,
1.2 equiv), DCC (1.08 g, 5.24 mmol, 1.3 equiv), HOBt (0.71 g,
5.24 mmol, 1.3 equiv), and dry DMF (35 mL) was stirred at room
temperature, under an argon atmosphere, during 30 min. After that
time, 1-naphthylamine (0.58 g, 4.03 mmol) was added and the
resulting reaction mixture was allowed to stir at room temperature
for 24 h. Upon filtration and removal of the solvent in vacuo, the
residue was purified by flash chromatography using as eluent
a mixture of chloroform/methanol (95:5) to afford 4 (512 mg, 31%) as
a white solid. (b) Using microwave irradiation: A mixture of 3-ben-
zyloxy-1-(30-carboxypropyl)-2-methyl-4-pyridinone 3 (63.1 mg,

A.M.G. Silva et al. / Tetrahedron 66 (2010) 8544e8550
8549
0.22 mmol, 1.2 equiv), DCC (49.1 mg, 0.24 mmol, 1.3 equiv), HOBt
(32.1 mg, 0.24 mmol, 1.3 equiv), 1-naphthylamine (26.2 mg,
0.18 mmol), and DMA (2 mL) was placed in a 10 mL reaction vial,
which was then closed under an argon atmosphere and placed in the
cavity of a CEM microwave reactor. The reaction vial was irradiated
at 55 ꢀC (1 min ramp to 55 ꢀC and 60 min hold at 55 ꢀC, using 100 W
maximum power). The reaction mixture was then purified by flash
chromatography using a 95:5 mixture of chloroform/methanol as
eluent to give 4 (22.6 mg, 30% yield). [Found: C, 73.29; N, 6.84; H
6.30. C26H24N2O3$2/3H2O requires C, 73.57; N, 6.60; H 6.02%]; dH
(400 MHz; DMSO-d6) 2.31 (3H, s, CH3), 2.92 (2H, t, J 6.8 Hz,
CH2CH2CONH), 4.27 (2H, t, J 6.8 Hz, CH2CH2CONH), 5.03 (2H, s,
CH2C6H5), 6.17 (1H, d, J 7.6 Hz, 5-H), 7.29e7.38 and 7.41e7.44 (5H,
2m, CH2C6H5), 7.47e5.56 (3H, m, naph-H), 7.62 (1H, d, J 7.2 Hz, naph-
H), 7.63 (1H, d, J 7.6 Hz, 6-H), 7.77 (1H, d, J 8.0 Hz, naph-H), 7.91e7.95
(2H, m, naph-H), 10.08 (1H, s, NH). dC (100 MHz; DMSO-d6) 12.0
(CH3), 36.6 (CH2CH2CONH), 49.3 (CH2CH2CONH), 71.9 (CH2C6H5),
116.2 (C-5), 121.9, 122.7, 125.5, 125.6, 125.9, 126.1, 127.7, 127.8, 128.2,
128.28, 128.34, 133.2, 133.7, 137.9, 139.7 (C-6), 140.7 (C-2), 145.5 (C-
3), 168.8 (CH2CH2CONH), 171.0 (C-4). m/z (MS, FAB) 413 [MþH]þ.
amount of the ligands solution in water (% DMSO less than 1%)
and with ionic strength 0.1 M in NaCl. Spectrophotometric pH
titrations were performed in stock aqueous solutions of the
ligands (w2.5ꢂ10ꢃ5 M), and aliquots of strong acid or base were
added to adjust pH to the desired value. Calculations were
performed with the program PHAB using data sets of at least
three independent experiments. A typical experiment includes
more than 10 solutions in which 10 different pH values have
been fixed.
4.8. Fluorescence spectroscopy
Steady-state fluorescence measurements were carried out in
a Varian spectrofluorometer, model Cary Eclipse, equipped with
a constant-temperature cell holder (Peltier single cell holder).
Stock solutions of the compounds were obtained by dissolution in
DMSO. All solutions were prepared by dilution of the right amount
of the compound solution in the solvent (% DMSO less than 1%). All
spectra were recorded with a lexc¼290 nm and emission between
300 and 550 nm for the ligands L1 and L2 and lexc¼325 nm and
emission between 350 and 550 nm for the naphthylamine. The slit
width values used for excitation and emission were 10 nm for L1
and L2 in DMSO and 5 nm for L1 and L2 in ACN. Fluorescence
quenching studies were achieved by successive addition of a con-
4.6. Synthesis of 1-(N-naphthylcarbamoylpropyl)-3-hydroxy-
2-methyl-4-pyridinone hydrochloride (L2)
A mixture of 4 (0.30 g, 0.73 mmol) and a catalytic amount of 10%
Pd/C (w/w) in ethanol (10 mL) and HCl (0.05 mL) was stirred under
a hydrogen atmosphere at room temperature for 5 h. The reaction
mixture was filtered through Celite and the solvent evaporated in
vacuum to give the crude product. The resulting residue was
crystallized in methanol/acetone to give the hydrochloride salt of
L2 (0.17 g, 65%) as a white powder. Dihydroxypyridinium form mp
224e226 ꢀC. [Found: C, 63.35; N, 7.87; H, 5.14. C19H18N2O3$HCl
requires C 63.60, N 7.81, H 5.34%]; dH (400 MHz; DMSO-d6) 2.65 (3H,
s, CH3), 3.16 (2H, t, J 6.7 Hz, CH2CH2CONH), 4.72 (2H, t, J 6.7 Hz,
CH2CH2CONH), 7.34 (1H, d, J 7.0 Hz, 5-H), 7.47e7.55 (3H, m, naph-
H), 7.60 (1H, d, J 7.2 Hz, naph-H), 7.77 (1H, d, J 8.2 Hz, naph-H),
7.86e7.94 (2H, m, naph-H), 8.28 (1H, d, J 7.0 Hz, 6-H), 9.57e11.42
(2H, br, OH), 10.25 (1H, s, NH). dC (100 MHz; DMSO-d6) 14.0 (CH3),
37.2 (CH2CH2CONH), 53.9 (CH2CH2CONH), 112.0 (C-5), 123.3, 124.0,
126.87, 126.93, 127.2, 127.4, 129.1, 129.5, 134.4, 135.0, 140.0 (C-6),
143.0 (C-2), 144.4 (C-3), 160.3 (C-4), 169.7 (CH2CH2CONH). m/z (MS,
FAB) 323 [MþH]þ.
stant volume (10 ml) of nitrate metal solutions to the cuvette (final
concentration range 0.0e1ꢂ10ꢃ5 M) containing a constant amount
of ligand (w10ꢃ6 M).
4.9. X-ray crystallography
Crystals of L1 and L2 with suitable quality for single crystal
X-ray determination were grown up from chloroform/methanol
solution.
Crystal data of L1: C16H14NO2Cl, M¼287.73, orthorhombic, space
group Pbcn, Z¼8, a¼8.7433(2), b¼13.1065(4), c¼24.0925(7) Å,
V¼2760.86(13) Å3,
r
(calcd)¼1.384 Mg mꢃ3
,
m
¼0.277 mmꢃ1. 25,774
reflections were collected and subsequently merged to 3372 unique
reflections with a Rint of 0.0509. The final refinement of 190 pa-
rameters converged to final R and Rw indices R1¼0.0516 and
wR2¼0.1006 for 2374 reflections with I>2
s
(I) and R1¼0.0870, and
wR2¼0.1116 for all hkl data.
Crystal data of L2: C19H19N2O3Cl, M¼358.81, monoclinic,
space group P21, Z¼2, a¼8.2793(5), b¼7.1661(5), c¼14.3206(9) Å,
The hydrochloride salt of L2 (100 mg; 0.28 mmol) was dissolved
in a (10:1) mixture of chloroform/methanol and washed twice with
a saturated aqueous solution of Na2CO3. Then the organic layer was
extracted with chloroform and dried over anhydrous Na2SO4. The
solvent was evaporated in vacuum and the resulting residue was
crystallized in chloroform/hexane to give quantitatively L2 in keto
form. Keto form mp 198e200 ꢀC dH (400 MHz; DMSO-d6) 2.41 (3H, s,
CH3), 3.99 (2H, t, J 6.8 Hz, CH2CH2CONH), 4.35 (2H, t, J 6.8 Hz,
CH2CH2CONH), 6.23 (1H, d, J 7.2 Hz, 5-H), 7.47e7.56 (3H, m, naph-
H), 7.60 (1H, d, J 7.2 Hz, naph-H), 7.65 (1H, d, J 7.2 Hz, 6-H), 7.77 (1H,
d, J 8.4 Hz, naph-H), 7.89e7.94 (2H, m, naph-H), 10.18 (1H, s, NH). dC
(100 MHz; DMSO-d6) 11.5 (CH3), 36.6 (CH2CH2CONH), 49.5
(CH2CH2CONH), 110.6 (C-5), 121.9, 122.8, 125.5, 125.8, 126.0, 127.7,
128.0, 133.1, 133.6, 137.9 (C-6), 145.3 (C-3), 168.8 (C-4 and
CH2CH2CONH).
V¼846.69(9) Å3,
b
¼94.782 (4)ꢀ,
r
(calcd)¼1.407 Mg mꢃ3
,
m
¼0.247 mmꢃ1.10,581 reflections were collected, and subsequently
merged to 4335 unique reflections with a Rint of 0.0264. The final
refinement of 237 parameters converged to final R and Rw indices
R1¼0.0535 and wR2¼0.1331 for 3943 reflections with I>2
s(I) and
R1¼0.0601, and wR2¼0.1383 for all hkl data.
The X-ray data were collected on a CCD Bruker APEX II at 150(2)
K using graphite monochromatized Mo K
a
radiation (l¼0.71073 Å).
The crystals of L1 and L2 were positioned at 35 mm from the CCD
and the spots were measured using a counting time of 60 s. Data
reduction including a multi-scan absorption correction were car-
ried out using the SAINT-NT from Bruker AXS. The structures were
solved by direct methods using SHELXS-97.17 and refined through
full-matrix least squares with SHELXL-97.18 Anisotropic thermal
parameters were used for all non-hydrogen atoms. The CeH
hydrogen atoms were introduced in the refinement at calculated
positions giving thermal parameters equivalent 1.2 times those of
the atom to which were attached. The NeH and OeH hydrogen
positions in L1 and L2 were found from the last Fourier maps and
they were included in the structure refinement with individual
isotropic temperature factors. Molecular diagrams were drawn
with PLATON.19 CCDC 753940 for L1, CCDC 753939 for L2 and CCDC
753941 for derivative 4 contain the supplementary crystallographic
4.7. Spectrophotometric determination of acidity constants
Electronic absorption spectra were recorded with a Varian
Cary bio50 spectrophotometer, equipped with a Varian Cary
single cell Peltier accessory, using quartz cells with 1 cm path
length, thermo stated at 25 ꢀC. Stock solutions of the ligands
were prepared by dissolution in DMSO. Acidity constants of both
ligands were obtained in aqueous solution by dilution of the right

8550
A.M.G. Silva et al. / Tetrahedron 66 (2010) 8544e8550
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data for this paper. These data can be obtained free of charge from
the Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/datarequest/cif.
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Acknowledgements
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Financial support from FCT through project PTDC/QUI/67915/
2006 is gratefully acknowledged. Andreia Leite (SFRH/BD/30083/
2006) thanks FCT for Ph.D. grant. We also thank Dr. Andrea Carneiro
from CENTI, V.N. Famalicão, for making available to us a CEM Dis-
cover microwave reactor. The Bruker Avance II 400 spectrometer is
part of the National NMR network and was purchased under the
framework of the National Programme for Scientific Re-equipment,
contracREDE/1517/RMN/2005, with funds from POCI 2010 (FEDER)
and (FCT).
10. Gonçalves, M. S. T. Chem. Rev. 2009, 109, 190 and references therein.
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Supplementary data
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White, A. G.; Baumes, J. M.; Smith, B. D. Chem. Commun. 2010, 46, 1068; (b)
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The supplementary data contains selected NMR spectra (1H, 13C,
COSY, HSQC, and HMBC) of derivative 2 and ligand L1, X-ray single
crystal structure of derivative 4, distribution diagrams as a function
of pH of both ligands L1 and L2 and the fluorescence spectra of L1 in
DMSO. Supplementary data associated with this article can be
found in the online version, at doi:10.1016/j.tet.2010.08.065.
€
(d) Abdel-Hamid, M. K.; Bremner, J. B.; Coates, J.; Keller, P. A.; Milander, C.;
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15. NMR tubes in acetonitrile-d3 and methanol-d4 were prepared from a saturated
solution of ligand L1 in DMSO-d6.
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