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Â. Novais et al.
BioorganicChemistry79(2018)341–349
of the most studied ligands, currently in clinical use as an oral for-
mulation for treatment of systemic iron overload occurring in hema-
tologic disorders such as β-Thalassemia or sickle cell anemia [9]. It has
also been reported that iron chelators have a therapeutic benefit in
many other disorders where iron seems to play a role in disease pro-
gression such as cardiovascular disease and neurodegenerative diseases
[8,9]. Siderophores have also been used in synthetic constructs (side-
romycins) combined with antibiotics to improve delivery of molecules
[19]. The fluorescent bidentate and hexadentate ligands were prepared
by coupling of the fluorophore (F2, F7, F8 or F20) to: i) a protected 3,4-
HPO bidentate ligand or ii) a protected 3,4-HPO hexadentate chelator,
in order to yield protected forms of fluorescent bidentate and hex-
adentate chelators, respectively. The final chelators were obtained by
deprotection under BCl3 of the protected forms according to experi-
mental procedures described elsewhere [16,20,21]. The protected bi-
dentate or hexadentate units were synthesized in our laboratory fol-
lowing previously published procedures [21,22].
The antimicrobial activity of a set of 3-hydroxy-4-pyridinones (3,4-
HPO) iron chelators has been previously evaluated but in only a limited
number of strains from few bacterial species, and the variable anti-
bacterial effect has been associated with the presence of different
substitutions [12–15]. In addition, a set of rhodamine-derived 3,4-HPO
iron chelators had previously been shown to inhibit the growth of
Mycobacterium avium [16–18], paving the way to ascertain the potential
inhibitory effect of these and other fluorescent chelators in other bac-
terial species.
In the present work, we report the synthesis and characterization of
a new fluorescent 3,4-HPO iron chelator functionalized with a car-
boxyrosamine fluorophore (MRB20). Its antibacterial activity was as-
sessed against a set of strains from diverse Gram-positive and Gram-
negative species, including multidrug resistant bacteria, and further-
more compared with that observed for a set of structurally related
chelators including Deferiprone.
The non-fluorescent bidentate (Hheepp) and hexadentate (CP256)
3,4-HPO chelating units used were produced by our research group
according to methods previously described [21,23]. The fluorescent
bidentate (MRB2, MRB7, MRB8) and hexadentate (MRH7 and MRH8)
chelators were synthesized previously according to methods described
in the literature and are in stock in our laboratory [16,20]. Chelator
MRB20 was newly synthesized in this study, as described below. A
diagram of the precursors (protected 3,4-HPO units and fluorophores)
used and the fluorescent and non-fluorescent chelators studied are
outlined in Fig. 1. The structural features of the different chelators in-
cluded in this study are summarized in Table S1, that may be relevant
to establish structure-activity relationships (SAR) for the 3,4-HPO iron
(III) chelators studied.
2.1.3. Synthesis of chelator MRB20
The new fluorescent ligand MRB20 was prepared using a straight-
forward synthetic protocol [16,17] involving the condensation of car-
boxyrosamine (F20) and 1,6-dimethyl-2-aminomethyl-3-benzyloxy-4-
pyridinone, using DCC and NHS coupling agents, in anhydrous DMF
(MRB20p). The protected chelating unit, 1,6-dimethyl-2-aminomethyl-
3-benzyloxy-4-pyridinone, was prepared from the commercially avail-
able kojic acid (5-hydroxy-2-hydroxymethyl-4-pyrone) by methods
described in the literature [16,21,24] (for the detailed synthetic ap-
proach please see Scheme S1 in supporting information). The synthesis
involved the preparation of the chlorokojic acid, followed by reduction
with zinc dust and HCl, condensation with formaldehyde and benzy-
lation of the 3-hydroxyl group, to give 2-hydroxymethyl-3-benzyloxy-6-
methyl-4-pyrone. The 2-hydroxymethyl group was subsequently pro-
tected with 3,4-dihydro-2H-pyran, which after reaction with methyla-
mine and selective deprotection of the pyran group under mild acid
conditions, gave 1,6-dimethyl-2-hydroxymethyl-3-benzyloxy-4-pyr-
idinone. Subsequent conversion into the corresponding phthalimido
derivative and reaction with hydrazine, afforded the protected bi-
dentate unit having the NH2CH2- group in the position 2 of the ring.
The coupling reaction of the protected bidentate unit with the activated
carboxyl group of the carboxyrosamine produced MRB20p, which after
debenzylation with BCl3 in dichloromethane, furnished the desired
MRB20, as it is summarized in Scheme 1 and detailed below.
2. Material and methods
2.1. Chemistry
2.1.1. General information
Chemicals were obtained from Sigma–Aldrich (grade puriss, p.a.)
and were used as received unless otherwise specified.
NMR spectra were recorded on a Bruker Avance III 400, operating at
400.15 MHz for 1H and 100.62 MHz for 13C atoms, equipped with pulse
gradient units, capable of producing magnetic field pulsed gradients in
the z-direction of 50.0 G/cm or on a Bruker Avance III Two-dimensional
1H/1H correlation spectra (COSY), gradient selected 1H/13C hetero-
nuclear single quantum coherence (HSQC) and 1H/13C heteronuclear
multiple bond coherence (HMBC) spectra were acquired using the
standard Bruker software. NMR and Mass Spectrometry analyses were
performed at “Laboratório de Análise Estrutural, Centro de Materiais da
Universidade do Porto” (CEMUP) (Portugal). Elemental analyses were
performed at the analytical services of Universidad de Santiago (Spain).
Absorption spectra were acquired in a Shimadzu spectrophotometer
equipped with a constant-temperature cell holder, at 25 °C, in 1 cm
cuvettes.
Fluorescence measurements for photochemical characterization of
the new chelators were performed with
a
Varian Cary Elipse
2.1.3.1. Compound MRB20p. The 1,6-dimethyl-2-aminomethyl-3-
Spectrofluorometer equipped with a constant-temperature multicell cell
holder, at 25 °C, in 1 cm cuvettes. To minimize reabsorption effects, the
absorbance’s sample values were kept below 0.1. Flash chromatography
was carried out using silica gel Merck (230–400 mesh).
benzyloxy-4-pyridinone
(47.4 mg;
1.83 × 10−4 mol),
N,N′-
dicyclohexylcarbodiimide (DCC) (45.8 mg, 2.20 × 10−4 mol) and N-
hydroxysuccinimide (NHS) (25.5 mg, 2.20 × 10−4 mol) were added to
a solution of carboxyrosamine (F20) (97.0 mg, 2.20 × 10−4 mol) in
anhydrous DMF (1mL) and the mixture was stirred at room temperature
in the dark and under an argon atmosphere, for 2 days. Subsequently,
the N,N-dicyclohexylurea (DCU) precipitate formed was filtered off and
the solvent removed under reduced pressure. The product was purified
by gradient flash column chromatography, eluted with chloroform/
methanol (9:1), followed by an increase of the methanol rate until 8:2,
to afford MRB20p (0.0627 g; 49%) as a purple solid (Fig. 2).
2.1.2. Synthesis of 3,4-HPO chelators
The commercially available deferiprone® (also known as 1,2-di-
methyl-3-hydroxy-4-pyridinone, L1, CP20 or Hdmpp) and eight iron
chelator derivatives functionalized or not with fluorophores from the
rhodamine family, bearing different denticity (bidentate and hex-
adentate ligands) and functional groups were tested. According to
previous results [17], the stoichiometry of the reaction with iron was
taken into account. Four different rhodamine derivatives were con-
sidered as fluorophores: (a) sulforhodamine B (F2), (b) rhodamine B
isothiocyanate (F7), (c) carboxyrhodamine (F8) and (d) carboxyr-
osamine (F20) (Fig. 1). All the fluorophores are commercially available
with the exception of carboxyrosamine, previously synthesized by us
400.15 MHz 1H NMR (MeOD-d4, ppm): δ 1.31 (t, J 7.2 Hz, 12H,
NCH2CH3); 2.45 (s, 3H, 6″-CH3); 3.69 (m, 8H, NCH2CH3); 3.72 (s, 3H,
NCH3); 4.73 (s, 2H, CH2NH); 5.22 (s, 2H, CH2C6H5); 6.50 (s, 1H, H-5″);
6.99 (d, J 2.5 Hz, 2H, H4+H5); 7.06–7.09 (dd, J 2.5 and J 12.0 Hz, 2H,
H2+H7); 7.27–7.36 (m, 5H, CH2C6H5); 7.45–7.48 (d, J 12.0 Hz, 2H,
H1+H8); 7.58 (d, J 8.5 Hz, 2H, H2′+H6′); 8.08 (d, J 8.5 Hz, 1H,
342
Novais, ?ngela
