Hydroxypyridinones with enhanced iron chelating properties. Synthesis, characterization and in vivo tests of 5-hydroxy-2-(hydroxymethyl)pyridine- 4(1H)-one

Article abstract of DOI:10.1039/c6dt00129g

The synthesis of 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one (P1) is presented, together with the evaluation of its coordination ability towards Fe3+, studied by a combination of chemical, computational, and animal approaches. The use of complementary analytical techniques has allowed us to give evidence of the tautomeric changes of P1 as a function of pH, and to determine their influence on the coordinating ability of P1 towards Fe3+. The pFe3+ value 22.0 of P1-iron complexes is noticeably higher than that of deferiprone (20.6), one of the three clinical chelating agents in therapeutic use for iron overload diseases. This is due on one side to the tautomeric change to the catechol form, and on the other to the lower protonation constant of the OH group. Bio-distribution studies on mice allowed us to confirm in vivo the efficacy of P1. Furthermore the coordinating ability toward Al3+, Cu2+ and Zn2+ has been studied to evaluate the possible use of P1 against a second toxic metal ion (Al3+), and to envisage its potential influence on the homeostatic equilibria of essential metal ions. The chelating ability of P1 toward these ions, not higher than that of the corresponding deferiprone, contributes to render P1 a more selective iron chelator.

Full text of DOI:10.1039/c6dt00129g

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Gano, Z. Szewczuk, M. A. Zoroddu, M. Peana, A. Dominguez-Martin and D. Choquesillo-Lazarte, Dalton
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Hydroxypyridinones with enhanced iron chelating properties.
Synthesis, characterization and in vivo tests of 5-hydroxy-2-
(hydroxymethyl)pyridine-4(1H)-one.
Received 00th January 20xx,
Accepted 00th January 20xx
J. I. Lachowicz,a V. M. Nurchi,a G. Crisponi,a M. G. Jaraquemada-Pelaez,a M. Arca,a A. Pintus,a M. A.
Santos,b C. Quintanova,b L. Gano,c Z. Szewczuk,d M. A. Zoroddu,e M. Peana,e A. Domínguez-
Martín,f and D. Choquesillo-Lazarte,g
DOI: 10.1039/x0xx00000x
www.rsc.org/
The synthesis of 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one (P1) is presented, together with the evaluation of its
coordination ability towards Fe3+, studied by combining chemical, computational, and animal approaches. The use of
complementary analytical techniques has allowed to give evidence of the tautomeric changes of P1 as a function of pH,
and to determine their influence on the coordinating ability of P1 towards Fe3+. The pFe3+ value 22.0 of P1-iron complexes
is noticeable higher than that of deferiprone (20.6), one of the three clinical chelating agents in therapeutic use for iron
overload diseases. This is due on one side to the tautomeric change to the catechol form, and on the other to the lower
protonation constant of the OH group. Bio-distribution studies on mice allowed to confirm in vivo the efficacy of P1.
Furthermore the coordinating ability toward Al3+, Cu2+ and Zn2+ has been studied to evaluate the possible use of P1 against
a second toxic metal ion (Al3+), and to envisage its potential influence on the homeostatic equilibria of essential metal ions.
The chelating ability of P1 toward these last ions, not higher than that of the corresponding deferiprone, contributes to
render
P1
a
more
selective
iron
chelator.
ability of DFP to mobilize the excess of iron in thalassemic
patients. Hydroxypyridinones share common features with
catechols and hydroxamic acids.2-3 The zwitterionic aromatic
resonance form of deprotonated hydroxypyridinones is
isoelectronic with the catecholate dianion.4 This family of
ligands, encompassing three subfamilies, 1-hydroxy-2(1H)-
pyridinone (1,2-hopo), 3-hydroxy-2(1H)-pyridinone (3,2-hopo)
and 3-hydroxy-4(1H)-pyridinone (3,4-hopo) (Scheme 1), forms
five-member chelate rings in which the metal ion is bound by
two oxygen atoms.4
There are more than 4968 publications related to DFP in the
literature (SciFinder 21st December 2015). Different DFP
derivatives have been synthesized and characterized, the
majority of which involve substitutions in positions 1 and 2
with aliphatic and aromatic groups aimed at improving the
chelating properties, changing the lipophilicity and the
solubility, or gaining additional properties such as
fluorescence. 1’-Hydroxyalkyl substituents in position 2 lead to
a reduction of protonation and iron complex formation
constants, as compared to those of simple alkyl-substituted
hopos. This results from the combined effect of intramolecular
hydrogen bonding, between the 2-(1’-hydroxyl) group and the
adjacent 3-hydroxyl function, and also the negative inductive
effect of the 2-(1’-hydroxyl) group from the pyridinone ring,
stabilizing the ionized species. Although such an effect reduces
the overall stability constants for Fe3+, it also reduces the
proton affinity of the chelating functions. These changes result
in an increase in the corresponding pFe3+ values at pH 7.4.
Introduction
Hydroxypyridinone ligands have been extensively studied as
candidates for orally active iron chelators since the 1980s. The
European Union approved 3-hydroxy-1,2-dimethylpyridin-
4(1H)-one (deferiprone, DFP) (Scheme 1) as a clinical chelating
agent for iron overload diseases in 2000. A major drawback of
DFP is that it undergoes extensive metabolism in the liver. 3-
hydroxy functionality, crucial for iron scavenging, is the prime
target of this metabolism. Urinary recovery studies in rats and
humans showed that >44% and >85% of the administered dose
is recovered as the non-chelating 3-O-glucuronide conjugate
respectively.1 This extensive biotransformation limits the
a.Department of Chemical and Geological Science, University of Cagliari, Cittadella
Universitaria, 09042 Monserrato-Cagliari, Italy, E-mail: lachowicz@unica.it; Fax:
+39 070 6754478; Tel: +39 070 6754471
b.Centro Quimica Estrutural, Instituto Superior Tecnico, Universidade de Lisboa, Av.
Rovisco Pais, 1049-001 Lisboa, Portugal
c. Campus Tecnológico e Nuclear, Instituto Superior Técnico, Universidade de
Lisboa, Estrada Nacional 10, 2695-066 Bobadela LRS, Portugal
d.Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw,
Poland
e. Dipartimento di Chimica e Farmacia, Università di Sassari, Via Vienna 2, 07100
Sassari, Italy
f. Department of Inorganic Chemistry, Faculty of Pharmacy, Campus Cartuja,
University of Granada, E-18071, Granada, Spain
g.Laboratorio de Estudios Cristalográficos, IACT, CSIC-Universidad de Granada, Av.
de las Palmeras 4, E-18100, Armilla, Granada, Spain
†Electronic
Supplementary
Information
(ESI)
available.
See
DOI: 10.1039/x0xx00000x
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Conversely, the introduction of a hydroxyl function in position B.V and desferal from Biofutura Pharma. All the reagents were
View Article Online
DOI: 10.1039/C6DT00129G
2 does not induce similar effects, and instead results in a used without any further purification. Carbonate-free 0.1 M
decrease in lipophilicity as compared to the simple alkyl KOH solution was prepared as previously reported.9 The metal
derivatives.1 An additional increment is also observed with 6- ion standard solutions were prepared by dissolving the
alkyl substitution.1 Generally, N-alkylation of 3,4-hopos results required amount of chloride salts in pure double distilled
in little change in the affinity for protons as well as for Fe3+.5, 6 water and adding a stoichiometric amount of HCl to prevent
Surprisingly, only a few derivatives with hydrogen in position 1 hydrolysis.
Fe3+
solution
was
standardized
by
are known; 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one, spectrophotometric analysis of the Fe3+-desferal complex,
with its tautomeric forms, has been studied by computational while Al3+, Cu2+ and Zn2+ solutions were standardized by EDTA
methods, but not accompanied by experimental studies.7 titration.
Although different authors have debated about tautomeric 5-Benzyloxy-2-(hydroxymethyl)-4H-pyran-4-one
equilibrium of the free DFP ligand and its derivatives, no
Sodium hydroxide aqueous solution 11 M (10 mL) was added
tautomeric equilibria in the corresponding metal complexes
to 100 mL of a solution of KA (14.2 g, 100 mmol) in MeOH and
have been experimentally proven.
the mixture was heated under reflux. Benzyl chloride (12.75
Tautomers are usually characterized by different
mL, 111 mmol) was added drop-wise, and the reaction mixture
hydrophobicity, pK and structure, and therefore different
was refluxed overnight. Once the reaction finished (checked by
coordination ability. In this respect, the influence of the
TLC; eluent: DCM/MeOH, 1:0.75), the mixture was cooled to
medium polarity may be highly significant since a biological
room temperature. The precipitate was filtered and washed
system involves either an aqueous medium (blood or plasma),
with an ethylic ether/petroleum ether mixture to obtain a light
or an essentially non-protic medium such as a cell membrane
brown solid (18.016 g, 77% yield); M.p. 129 -131 °C (lit. value
or even an enzymatic reaction centre.8 Based on these
1
129-130°C10 and 132°C11). H NMR (300 Hz, MeOD), δ (ppm):
concerns, we considered that metal coordination may be
improved in 3,4-hopo with a N-H group in position 1, and
hence in this work we present a study on the coordination
8.01 (s, 1H, 6-HPy), 7.44–7.32 (m, 5H, HPh), 6.51 (s, 1H, 3-HPy),
5.02 (s, 2H, CH2-Bz), 4.41 (s, 2H, CH2-OH).
5-Benzyloxy-2-(hydroxymethyl)-pyridin-4(1H)-one
ability of 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one (P1)
(Scheme 1) towards Fe3+ by combining computational,
5-Benzyloxy-2-(hydroxymethyl)-4H-pyran-4-one (6.25 g, 26.91
mmol) was dissolved in methanol (25 mL) and then 25%
chemical, and biological approaches. The use of
ammonia aqueous solution (57.5 mL) was added. The mixture
complementary analytical techniques has allowed to give
was refluxed in oil bath for 7 h and the progress of the reaction
evidence of the tautomeric changes of P1 as a function of pH,
was monitored by TLC (eluent: DMC/MeOH 6:0.75 solvent
and to determine all of their possible forms and properties.
The coordinating ability of P1 towards Fe3+ together with bio-
mixture). The reaction mixture was concentrated in a rotary
evaporator to obtain a pale brown solid residue, which was re-
crystallized from ethanol/ethyl ether, leaving the pure target
distribution studies on mice to evaluate in vivo its efficacy is
also presented. Furthermore the coordinating ability toward
Al3+, Cu2+ and Zn2+ has been studied to evaluate the possible
compound as beige product (5.659 g, 90% yield); M.p. 224 °C
1
(lit. value 225-226 °C10). H NMR (300 Hz, DMSO), δ (ppm):
use of P1 against a second toxic metal ion (Al3+), and to
11.3 (s, 1H, NH), 7.42 -7.30 (m, 5H, HPh), 6.11 (s, 1H, 6-HPy),
envisage its potential influence on the homeostatic equilibria
5.70 (s, 1H, 3-HPy), 5.00 (s, 2H, CH2-Bz), 4.33 (s, 2H, CH2-OH).
of essential metal ions.
5-Hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one (P1)
5-Benzyloxy-2-(hydroxymethyl)-pyridin-4(1H)-one (2.542 g,
10.99 mmol) was dissolved in methanol (200 mL) and 10%
Pd/C (0.258 g) was added. The suspension was stirred for 4 h
under H2 atmosphere (2 bar). Afterwards the mixture was
filtered and the solvent evaporated. Final recrystallization
from EtOH/Et2O afforded the pure product a pale brown
1
colour (1.065 g, 70% yield); M.p. 245-247 °C. H NMR (300 Hz,
MeOD), δ (ppm): 7.48 (s, 1H, 6-HPy), 6.44 (1s, 1H, 3-H-Py), 4.55
(s, 2H, CH2-OH). 13C NMR (400 Hz, D2O), δ (ppm): 171.73
(C=O), 147.43 (C-OH), 145.65 (2-C-Py), 121.71 (6-C-Py), 111.09
Scheme 1. Hydroxypyridinone ligands and their acronyms.
(3-C-Py), 59.52 (CH2OH). Elemental analysis (%) calculated for
C6H7NO3: C 51.07; H 5.00, N 9.92%; found: C 50.53, H 5.12, N
9.65%.
Experimental section
Reagents
Analysis of the synthesized ligand
Elemental data were obtained using a Fisons EA1108 CHNS/O.
5-hydroxy-2-hydroxymethyl-pyran-4-one (Kojic acid, KA),
NMR spectra were recorded on a Bruker AscendTM 400 MHz
spectrometer. Chemical shifts (δ) were reported in ppm from
ammonia, benzyl chloride, NaOH, KOH, FeCl3, HCl solution, and
ethanol were purchased from Aldrich, KCl from Carlo Erba
the internal reference 3-trimethylsilyl-propionic acid-d4
(Milan, Italy), gallium citrate 67Ga from Mallinckrodt Medical
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sodium salt (DSS). The melting points were measured on a solutions (70 μL). Solutions were introduced at a flow rate of 3
View Article Online
DOI: 10.1039/C6DT00129G
Leica Galen III hot stage apparatus and are uncorrected.
Crystal Structure Determination
μL/min. The Bruker microTOF-Q spectrometer was calibrated
with the TunemixTM mixture (Bruker Daltonik, Germany) in
the quadratic regression mode. The mass spectrometer
operated in the positive ion mode. Each spectrum was
obtained with more than 100 individual scans. The overall
charge of the analysed ions was calculated on the base of
distance between isotopic peaks. The formulae of the
complexes were determined by application of the Compass
Data Analysis program (Bruker Daltonik, Germany).22 The
water and D2O solutions of the ligand (1×10-4 M) with FeCl3
and AlCl3 salts (1:3 metal to ligand molar ratio) and CuCl2, ZnCl2
(1:2 metal to ligand molar ratio) were incubated for 15 days at
room temperature before the measurements. Solutions at
variable pH and pD values were obtained by adding
triethylamine and HCl (water solution), or NaOD and DCl (D2O
solutions). pH and pD were measured on a previously
calibrated pH-meter.
Single crystals of P1 were grown by slow evaporation of a
methanol solution (30 mg in 3 mL at 50 ˚C). A yellowish crystal
of P1 was mounted on a glass fibre and used for data
collection. Yellowish crystals were prepared under inert
conditions immersed in perfluoropolyether as protecting oil
for manipulation. A suitable crystal was mounted on MiTeGen
Micromount, and this sample was used for data collection.
Crystal data were collected at 100(2) K, using a Bruker D8
Venture diffractometer (Mo K(α) radiation λ = 0.71073 Å). The
data were processed with APEX212 and corrected for
absorption using SADABS (transmissions factors: 0.7456 -
0.6959).13 The structure was solved by direct methods, which
revealed the position of all non-hydrogen atoms.14 These
atoms were refined on F2 by a full-matrix least-squares
procedure using anisotropic displacement parameters. All
hydrogen atoms were located in difference Fourier maps and
NMR spectroscopy
included as fixed contributions riding on attached atoms with NMR experiments were performed on a Bruker AscendTM 400
isotropic thermal displacement parameters 1.2 (C-H, N-H) or MHz spectrometer equipped with a 5 mm automated tuning
1.5 (O-H) times those of the respective atom. The absolute and matching broad band probe (BBFO) with z-gradients as
structure for the compound was chosen according to the Flack previously reported.23 In particular, all the NMR experiments
parameter.15 Criteria of a satisfactory complete analysis were were carried with ligand concentration 5 mM in D2O and H2O -
the ratios of "rms" shift to standard deviation less than 0.001 D2O (90% -10%) solutions at 25 °C in 5 mm NMR tubes. The
and no significant features in final difference maps. Atomic solution pH, corrected for the deuterium isotopic effect, was
scattering factors were taken from "International Tables for adjusted with diluted DCl and NaOD on a pH meter Crison
Crystallography".16 Molecular graphics were plotted using micro TT 2050 with an electrode Mettler Toledo InLab 422. The
PLATON17 and Olex2 programs.18 A summary of the crystal concentration of Fe3+ and Ga3+ ions was achieved by using a
data, experimental details and refinement results are listed in stock acidic deuterated aqueous solution of FeCl3 and GaCl3
Table S1. Crystallographic data have been deposited with the salts. The metal ions were added to P1 ligand solutions and the
Cambridge Crystallographic Data Centre, CCDC 1056172 for pH was then set to the right value just before the acquisition of
1
ligand P1. Copies of this results may be obtained free of charge the spectra. 2-D H-13C HSQC (heteronuclear single quantum
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, coherence) spectra were acquired by using a phase-sensitive
UK(fax: 44 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk or sequence employing Echo-Antiecho-TPPI gradient selection
www: http://www.ccdc.cam.ac.uk).
Potentiometric-spectrophotometric measurements
with a heteronuclear coupling constant JXH = 145 Hz, and
shaped pulses for all 180° pulses on f2 channel with decoupling
during acquisition; sensitivity improvement and gradients in
back-inept were also used.24-26 Relaxation delays of 2s and 90°
pulses of about 10 μs were applied in all the experiments.
Solvent suppression for 1D 1H, 2D 1H-1H TOCSY (total
correlation spectroscopy) and 1H-1H ROESY (rotating-frame
Overhauser effect spectroscopy) experiments were achieved
by using excitation sculpting with gradients. The spin-lock
mixing time of TOCSY experiment was obtained with
MLEV17.27 1H-1H TOCSYs were performed by using mixing
times of 60 ms. A combination of 1D, 2D TOCSY, HSQC and
ROESY experiments was employed to assign the signals of both
free and metal-bound ligand at different pH values as
previously reported for similar systems.23, 28-33 All NMR data
were processed by using TopSpin (Bruker Instruments)
software and analysed by Sparky 3.1134 and MestRe Nova 6.0.2
(Mestrelab Research S.L.) programs.
Protonation and complex-formation equilibria were carried
out as previously described, at ionic strength KCl 0.1M.19 The
working ligand concentration was 4.9×10-4 M. Complex
formation studies were performed at 1:1, 1:2, and 1:3
metal/ligand molar ratios. Combined potentiometric-
spectrophotometric measurements were done in the 200-400
nm spectral range for protonation equilibria and in the 400-
800 nm range for Fe3+ complexation, using 0.2 and 1 cm path
lengths, respectively. Complex formation equilibria with Al3+,
Cu2+ and Zn2+ were potentiometrically studied. Protonation
and complex formation data were analysed using
HyperQuad201320 and HypSpec21 programs.
ESI-MS analysis
All MS experiments were performed on a Bruker microTOF-Q
spectrometer (BrukerDaltonik, Bremen, Germany), equipped
with an Apollo II electrospray ionization source with an ion
funnel. The instrument parameters were: scan range m/z 250-
2000, dry gas – nitrogen, temperature 200 °C, ion source
voltage 4500 V, collision energy 10 eV, analyte aqueous
DFT calculation
Theoretical calculations were performed at Density Functional
Theory (DFT) level35 with the Gaussian 09 package of programs
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(Rev. A.02)36 on a E4 workstation equipped with four quad-
core AMD Opteron 2.2 MHz processors and 16 Gb of RAM and
running the 64 bit version of the Ubuntu 12.04 Linux operating
system. Barone and Adamo’s mPW1PW hybrid functional was
Results and discussion
View Article Online
DOI: 10.1039/C6DT00129G
Synthesis of P1
5-Hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one
was
adopted,37 along with the Schäfer, Horn, and Ahlrichs double-

synthesised from kojic acid, following a standard synthetic
procedure, previously described for the preparation of other
pyridinones.44 The first step involves the protection of the 5-
hydroxyl group of KA with a benzyl group, by reaction with
benzyl chloride and formation of 5-(benzyloxy)-2-
(hydroxymethyl)-4H-pyran-4-one. This was converted in the
corresponding pyridinone, 5-benzyloxy-2-(hydroxymethyl)-
pyridin-4(1H)-one, by reaction with an ammonia aqueous
solution under reflux (Michael addition, ring opening/closure,
and dehydration). The benzyl protecting group was then
removed by catalytic hydrogenolysis providing the final
product P1.
plus polarization all-electron basis sets38 (BS) for all atomic
species. The geometry of the compounds was optimized with
tight SCF convergence criterion and fine numerical “pruned”
integration grids (75 radial shells and 302 angular points per
shell). The nature of the minima of each optimized structure
was verified by harmonic frequency calculations. NBO charges
distributions39 and Wiberg40 bond indices were calculated at
the optimized geometries. Solvation calculations were carried
out in the gas phase and water, implicitly taken into account
by means of the polarisable continuum model (PCM) in its
integral equation formalism variant (IEF-PCM).41 The programs
Gaussview 5.0.8,42 and Molden 5.2.443 were used to
investigate the charge distributions and optimized geometries.
Partition coefficients
P1 and DFP octanol/water partition coefficients (log P) were
calculated as the logarithm of the ratio of their concentrations
at 25 °C measured in n-octanol phase and in aqueous phase
(Tris, pH = 7.4), using the well-established ‘‘shake flask”
method.10, 11, 44-46 Each compound was added to a premixed
suspension, containing 3 mL of n-octanol and 3 mL of the
aqueous phase, and stirred for 4 h. The species concentration
in the aqueous layer was evaluated by spectrophotometry
with a Perkin Elmer Lambda 35 spectrophotometer, using a
thermostated 1 cm path length cell, on the benzenoid bands of
the compounds (λ = 280–290 nm). The measurements were
done in triplicate and repeated three times.
Scheme 2. Synthesis of P1.
Crystal Structure of P1
The bond lengths and angles in the molecule P1 (Fig. 1) have
commonly encountered values (Table S3). The substituents at
C4 and C5 are almost coplanar with the aromatic ring. In
contrast, the torsion angle N1–C2–C7–O8 [-31.0(2)º] reveals a
noticeable twisting for the C2 hydroxymethyl group towards
the aromatic ring plane.
Biodistribution studies
67Ga-citrate injection solution was prepared by dilution of the
commercial 67Ga citrate solution with saline to obtain a final
radioactive concentration of 5 -10 MBq/100 L. Biodistribution
studies were carried out in groups of 3 female Balb-C mice,
weighing ca. 20-22 g (randomly bred, Charles River, from
CRIFFA, France). Mice were intravenously injected with 100
(5 -10 MBq) of 67Ga citrate via the tail vein immediately
followed by intraperitoneal injection of 0.35 mol of each
ligand (P1 and DFP as a control) in 100 L saline. Animals were
L
Fig. 1. Ball and stick structure and atom labelling scheme of P1.

The crystal packing of P1 features infinite one-dimensional zig-
zag chains along c axis, built up by classical hydrogen bonding
interactions: N1-H1AO4c and O5-H5AO8a (operators for

maintained on normal diet ad libitum and were sacrificed by
cervical dislocation at 1h and 24 h post-administration. The
administered radioactive dose and the radioactivity in
sacrificed animals were measured in a dose calibrator (Aloka,
Curiemeter IGC-3, Aloka, Tokyo, Japan). The difference
between the radioactivity in the injected and in sacrificed
animal was assumed due to the whole body excretion. Tissue
samples of main organs were then removed for counting in a
gamma counter (Berthold LB2111, Berthold Technologies,
Germany). Radioactivity remaining in the carcass was
measured in the dose calibrator and expressed as percent of
injected activity (%I.A.). Biodistribution results were expressed
as percent of injected activity per gram of organ (%I.A./g) and
presented as mean values ± SD.
generating equivalent atoms: a, -x+1/2, -y+1, z-1/2; c, -x+1/2, -
y+1, z+1/2, with with R hydrogen-bonding patterns (Fig.
2B).47
Fig. 2. Crystal packing of P1 seen along the a (A) and b (B) directions.
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Each molecule forms an additional hydrogen bond O8- predominantly H7 signals of methylene group, which could be
View Article Online
H8AO4b (b, -x+1, y+1/2, -z+1/2) with a molecule of a implied in a hydrogen bond between tDheOI:O10H.10g3r9o/uCp6DaTn0d012th9Ge
neighbouring chain to give an infinite three-dimensional now deprotonated nitrogen atom. The high pK of this last
network (Fig. 2A). Hence, the O4-carbonyl atom acts twice as dissociation suggests a keto-enol tautomer transition with
H-acceptor whereas the O8-H8A alcoholic moiety plays both H- dissociation from the hydroxyl group.
donor and H-acceptor roles. The structural comparison of P1
Table 1. Protonation constants for P1 at 25°C, 0.1 M KCl ionic strength, obtained using
with kojic acid,48 shows that the O-heterocyclic atom of kojic
acid does not act as H-acceptor in its crystal. In clear contrast,
the isolobal N1-H1A group of P1 (replacing the O-heterocyclic
donor of the kojic acid) acts as an H-donor for intermolecular
interactions. In addition, the torsion angle O1–C2–C7–O8 [–
167.8(2)˚] in KA shows that the hydroxymethyl substituent is
oriented in the opposite direction respect to KA. These data
will be of importance in the following considerations of
tautomeric forms.
HyperQuad2013 program.20 Standard deviations on the last figure in brackets. The
literature values for DFP (Scheme 1, HL' in the neutral form) and 3,4-hopo are reported
for comparison.
P1
Log K
DFP
3,4-hopo
Log K4
Species
Species
Log K49
H3L+
H2L
HL–
3.37(7)
9.00(1)
12.16(1)
H2L'+
HL'
3.66(1)
9.82(2)
-
3.34(1)
9.01(1)
-
Protonation equilibria
The protonation equilibria of P1 have been examined with
different techniques. Potentiometric-spectrophotometric
measurements gave evidence of three different deprotonation
steps according to the sequence (1).
The log K 9.00 and 3.37 are almost equal to those of 3,4-hopo4,
and somewhat lower than those of DFP (Table 1).49
Conversely, the loss of a third proton is a new feature, which
implies an equilibrium with a catechol-like molecule that loses
its proton at basic pH values (log K 12.16). The deprotonation
at the ring NH group of DL-α-amino-β-(6-oxo-1,6-dihydro-
pyridin-6yl)propanoic acid (Stunzi et al.)50 is characterized by a
log K value of 11.8, similar to that of 2-hydroxypyridine
(11.7).51
The ESI-MS measurements in D2O give evidence of the
exchange of four labile protons with deuterium. The spectrum
of ligand in D2O solution (Fig. S9) shows five signals of different
intensities. The most intense signals at 145.070 m/z and
146.076 m/z correspond to [C6H5D3O3N]+ and [C6H4D4O3N]+
deuterated on two or three oxygen atoms and on a single
nitrogen atom. The less intense signals at 142.050, 143.057
and 144.063 m/z match with the simulated spectra of
[C6H8O3N]+, [C6H7DO3N]+ and [C6H6D2O3N]+. The ESI-MS signals
of free ligand at different pH values (1.68, 6.13, 11.5 and 13.7,
Table S28) show that the molecular weight of the ligand does
not change during the first (140.032 m/z) and third (139.032
m/z) deprotonation in negative mode spectrum, and does not
present positively charged signals at basic pH. The presence of
a peak at 139.032 m/z, a sticking peculiarity observed in
aqueous solutions at pH >11.5, further confirms the last
deprotonation process shown in Sequence 1.
H3L+ = H2L = HL– = L2– (1)
The loss of each proton markedly affects UV absorption
spectra (Fig. S2A and B), and three different pH regions (3 –
4.6, 7.3 – 10.5, and 10.6 – 13.2) relative to each single
equilibrium can be identified, characterized by different bands
and well defined isosbestic points. These features allowed the
evaluation of the protonation constants (Table 1); the related
speciation plot is presented in Fig. 3A with superimposed the
heights of UV bands, obtained by spectral decomposition. The
1
trend of H NMR signals of protons H3, H6 and H7 vs pH (Fig.
3B, Fig. S4 and Table S27) confirms the deprotonation
sequence (1).
The deprotonation sequence of P1 has been also investigated
at DFT level by calculating the electronic energies of the
possibly protonated forms of the ligand at their ground state
Fig. 3. A) Normalized heights of the peaks at 248 nm(blue), 273nm (red) and 307 optimized geometry. Based on the results recently published
on related systems, DFT calculations were performed by using
nm (violet), obtained by spectral decomposition, superimposed to the speciation
the mPW1PW hybrid functional37 and Schäfer, Horn, and
plot of ligand. B) Normalize chemical shifts of H6 (●), H3 (■) and H7 (Δ) proton,
Ahlrichs double- plus polarization all-electron basis sets (BS)
superimposed to the speciation plot of ligand.
for all of the atomic species.38 In particular, the fully
protonated cationic ligand H3L+, the three possible isomers of
the neutral and monoanionic forms (H2L and HL–, respectively),
and the completely deprotonated dianion L2– were optimized,
both in the gas phase and in water, implicitly taken into
account by using the integral equation formalism of the
polarizable continuous model (IEF-PCM) within the self-
consistent reaction field (SCRF) approach (Scheme 3).41
The first deprotonation (pH 2-5) mainly affects proton H3,
suggesting that the proton is lost from the protonated oxygen
in position 4. The signal of H6 reflects this effect though in a
minor extent. The second step (pH 7.3 - 10.5), related to the
deprotonation of the 5-hydroxy group, mainly reflects on H6
chemical shift. The last deprotonation ( pH > 10.6) affects
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H4, as hypothesized based on spectroscopic evVidieewnAcrteicsle,Oannlinde
DOI: 10.1039/C6DT00129G
with the X-ray diffraction analysis discussed above. In
agreement with experimental data, H2La is stabilized by a
hydrogen bond (O4···H5 distance = 1.885 Å; Fig. S1), the
interactions between the LP localized on O4 and the unfilled
natural orbitals of the O5-H5 hydroxyl group being calculated
in ESOPTA = 10.6 kcal/mol.
As far as the monoanionic forms of the ligand are concerned,
–
–
the isomers HLa and HLc were calculated to be more stable
than HL– by 12 and 15 kcal/mol, respectively, thanks to the
b
formation of two different hydrogen bonds, one involving the
N1 atom and the hydroxymethyl pendant, and the other the
C4–O4 and C5–O5 groups (O5···H4 distance in HLa– = 1.684 Å;
O4···H5 distance in HLc– = 1.775 Å; ESOPTA
27 and 18 kcal/mol
–
–
for HLa and HLc , respectively). This accounts for the
mechanism suggested by experimental data for the second
deprotonation of the ligand, which should involve the O5-H5
hydroxyl group, then followed by a rearrangement leading
Scheme 3. Lewis structures of the differently protonated forms of P1 optimized at the
DFT level.
–
from the pyridinone HLb– to the cathecol-like tautomer HLa .
Each technique has given evidence of tautomeric forms, and
A comparison of the metric parameters optimized for the together they provided
a complete description of the
neutral isomer H2La in the gas phase with the corresponding deprotonation paths. As long as third deprotonation was not
structural data (see above) shows very good agreement observed for 3,4-hopo, we believe that –CH2OH in the position
between the two sets of data, the principal bond distances 2 is important for the tautomer change of the molecule.
being varied by less than 0.05 Å (Table S7). No significant Iron complexes
changes in the optimized structural parameters were observed
The iron complex formation was studied by potentiometric-
upon considering implicit solvation in the calculations, all the
spectrophotometric combined titrations, ESI-MS and NMR.
Some spectra collected during a Fe3+-P1 titration are presented
bond lengths and angles differing by less than 0.01 Å and 2°,
respectively for all of the eight forms of the ligand considered
in Figure 5, representative of the transformations that occur
here. All of the compounds show a completely planar
with pH. Up to pH 2 a new band centred at 550 nm increases
in intensity (Fig. 4A), due to the formation of a [FeLH]2+
structure, except the hydroxymethyl substituent, whose
orientation depends on the protonation pattern of the ligand.
complex from the colourless hexaaquo-iron species. In the pH
In particular, in the species where N1 is deprotonated (H2Lb,
range 2-3 (Fig. 4B) a new band appears at 500 nm with a clear
–
–
HLa , HLc , and L2–), the O8-H8 hydroxyl group is coplanar with
isosbestic point at 585 nm, related to the formation of a
[FeL2H2]+ complex. By increasing the pH a new band forms at
460 nm related to the neutral [FeL3H3] complex, with an
isosbestic point at 488 nm. The behaviour up to pH 5 is similar
to that of deferiprone that presents in sequence similar bands
the heterocyclic ring, and H8 is oriented towards N1 (C2-C7-
O8-H8 dihedral angle
 0.0°), so that a hydrogen bond
interaction can be observed between the two atoms (N1···H8
distance in the range 1.663-1.907 Å; Fig. S1 for H2Lb). A
Second-Order Perturbation Theory Analysis (SOPTA) of Fock
matrix in NBO basis allows for evaluation of the energy ESOPTA
of the interaction between the lone pair (LP) localized on N1
and the unfilled natural orbitals of the O8-H8 hydroxyl group in
the range between 10.3 and 35.0 kcal/mol depending on the
species. On the other hand, in the forms of the ligand showing
due to the formation of 1:1, 1:2 and 1:3 complexes.49
A
different behaviour is featured by P1 at pH > 5: first a new
band appears at 445 nm with a clear isosbestic point at 464
nm, that at pH > 9 further transforms into a new band at
slightly higher wavelength, with an isosbestic point at 485 nm.
These last transformations, taking into account the
potentiometric results, are related to the loss of a proton from
the neutral species modelled as FeL3H3 (possibly corresponding
to Fe(HL)3) giving a negatively charged complex modelled as
[FeL3H2]–, and then of two further protons to give the [FeL3]3–
complex. Such changes were observed above pH 7 by Scarrow
et al. for 3,2-hopo, but not for 3,4-hopo.4 The first isosbestic
point of tris(3,2-hopo) iron complex at 494 nm was attributed
by the authors to the mono-deprotonation with pK value of
9.04. A second deprotonation occurred around pH 11. It has to
be remarked that, while DFP and P1 iron complexes are well
–
N1 protonated (namely H2La, H2Lc, and HLb ), in agreement
with what was found experimentally for H2La, the O8-H8
moiety lies outside the plane described by the rest of the
molecule, the N1-C2-C7-O8 and C2-C7-O8-H8 dihedral angles
being in the ranges –23.0/–40.8 and –49.2/–78.8°, respectively
(Table S7 and Fig. S1 H2La and H2Lc). Finally, in H3L+, although
the C7 hydroxyl group is coplanar with the heterocyclic ring,
H8 is oriented opposite to N1 (C2-C7-O8-H8 dihedral angle

180.0°). An analysis of the total electronic energies calculated
for the three possible neutral tautomeric forms of the ligand
suggests the tautomer H2La to be the most stable (H2Lb and
H2Lc being less stable by about 7 and 5 kcal/mol, respectively).
This agrees with a first deprotonation of the ligand concerning
soluble
in
the
pH
range
2-11,
the
neutral
tris(hydroxypyridinonato) iron complexes with the three hopo
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ligands studied by Scarrow et al.4 are quite insoluble. The
negative charge in the complexes of P1 with general model
View Article Online
DOI: 10.1039/C6DT00129G
[FeL3H3–n]
n– further contributes to increase their solubility.
The stability constants of the Fe3+-P1 complexes obtained by
the spectrophotometric-potentiometric results are reported in
Table 2, and the related speciation plot in Figure 5.
Fig. 5. Speciation plots of P1 complexes with Fe3+
10 µM ligand concentration (log β in Table 2).
The pFe3+ value 22.0 of P1-iron complexes is noticeable higher
than that of deferiprone (20.6),49 3,4-hopo (20.6)4 and 3,2-
hopo (16.3).4 This is due on one side to the loss of the nitrogen
proton from the complex with model FeL3H3, with
corresponding tautomeric change to the catechol form, and on
the other to the protonation constant of the OH group lower
in P1 than in deferiprone.
The
ESI-MS
data
confirm
the
potentiometric-
spectrophotometric results. Positively charged complexes form
at acidic pH: [FeL2H2]+ (336.004 m/z), [FeL2H2+Cl-+K+]+ (409.940
m/z) and [FeL3H3+K+]+ (515.002 m/z), while at neutral and
basic pH [FeL2H2]+ (336.004 m/z), [FeL3H3+Na+]+ (499.029 m/z)
and [FeL3H3+K+]+ (515.002 m/z), complexes are present. At
basic pH also, the formation of negatively charged complex
[FeL2]- (333.998 m/z) is observed (Fig. S10 and Table S29).
The interaction of P1 with Fe3+ ions has been followed by NMR
at two different pH values. In Figure 6 the aromatic region of
1H NMR spectra of P1 is reported. The spectra were obtained
by using sub-stoichiometric amounts of Fe3+ ions, from 0:1 till
to 0.2:1 metal to ligand molar ratio at pH 7 and 11.5. Both
aromatic protons, H3 and H6, are affected by the interaction
with Fe3+ ions.
Fig. 4. Spectra obtained by increasing pH values of Fe3+-P1 system (25˚C, 0.1 M
KCl ionic strength, P1 concentration 4.9 × 10-4 M, 1:3 Fe/L molar ratio, optical
path length 1 cm). These spectra were divided in different pH ranges to remark
the transformations there occurring.
1
Table 2. Metal-ligand complex formation constants at 25˚C, 0.1 M KCl ionic strength,
obtained using HyperQuad2013 program.20 Standard deviations on the last figure in
brackets.
In the aromatic region of H NMR spectra, at pH 7, a selective
broadening of signals was evidenced and both aromatic
protons, H3 and H6, were affected by the interaction with Fe3+
ions. In particular, the H6 proton is the most affected
suggesting the involvement in the coordination of the
deprotonated oxygen in the position 5 to give a FeL3H3 species.
Ga3+ ions have been used as a diamagnetic probe for the
paramagnetic Fe3+ ions, in order to prevent the NMR
Fe3+
Al3+
Model
Log
pK
Log
pK
[MLH]2+
[ML2H2]+
ML3H3
25.92(9)
49.12(1)
71.43(6)
65.33(3)
46.03(5)
23.32(9)
45.18(6)
65.25(7)
58.30(4)
38.41(5)
1
paramagnetic line broadening. The aromatic region of H NMR
[ML3H2]–
[ML3]3–
pM
6.1
9.65
6.95
9.95
spectra obtained by adding incremental amounts of Ga3+ ions
to P1 solution at neutral pH shows that, similarly to Fe3+, H6
and H3 were the most affected protons by metal coordination,
giving however in this case an upfield and a downfield shift,
respectively (Fig. S7).
22.0
15.1
Cu2+
Zn2+
Model
Log
pK
Log
18.79(6)
36.70(4)
26.02(9)
----
pK
[MLH]+
ML2H2
[ML2H]–
[ML2]2–
pM
21.78(7)
41.42(7)
34.21(5)
24.68(5)
The spectra obtained at pH 11.5 (Fig. 6) confirm that the
deprotonation of the pyridine nitrogen protons affects, after
Fe3+ coordination, in the same way H3 as well as H6,
suggesting the deprotonation of both oxygen atoms at pH > 11
without any hydrolysis of the complex. These features are in
7.21
9.53
10.68
10.1
6.3
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agreement with the proposed scheme of tautomerism.
On passing from the gas phase to the optimization in water
carried out at SCRF level, only minor variations in the pattern
View Article Online
DOI: 10.1039/C6DT00129G
of the bond lengths and angles were calculated (average Fe–O
bond distance: 2.036; C–O: 1.287 Å).
Fig. 6. Stacked aromatic region of 1D 1H NMR spectra of P1 by increasing sub-
stoichiometric amounts of Fe3+, at pH 7 (left) and pH 11.5 (right) at 25 °C.
For Ga3+-P1 system, the signals H3 and H6 were affected, also
at high pH, in a similar way to that of Fe3+-P1 (Fig. S8).
Some of the authors recently used DFT calculations to
investigate the relative stabilities of Al3+ and Fe3+ complexes
Fig. 7. Ball and stick drawing of the DFT-optimized molecular structure of the neutral
complex Fe(HL)3 (purple = Fe; red = O; grey = C; blue = N; white = H).
featuring
methoxyphenyl)methanediyl]bis
the
2,2'-[(2-hydroxy-3-
[3-hydroxy-6-
By complete deprotonation of the complex Fe(HL)3, the anionic
species [FeL3]3– is ideally obtained. As expected, the
coordination environment at the high-spin central metal ion is
not changed as compared to the corresponding neutral
complex, with only a marginal elongation of the Fe–O and C–O
distances [average optimised Fe–O distances: 2.056 Å; C–O:
1.287 Å; O–Fe–O, 78.77°; corresponding average structural
metric parameters for trianionic tris-catecholate Fe3+
complexes: Fe–O, 2.01(3) Å; C–O, 1.33(2) Å; O–Fe–O, 80(1)°],
reflecting an unchanged pattern of bond orders at the iron
centre [average Wiberg40 bond index: 0.332 and 0.331 for
FeL3H3 and [Fe(L)3]3–, respectively]. As observed for the free
ligand, the deprotonation of the N-heterocycles results in the
formation of hydrogen bonds between the nitrogen atoms and
the hydroxymethyl pendants (distance N···H(O) = 1.724 Å).
The SOPTA allows evaluating the energy of the non-bonding
hydrogen interaction in 13.1 kcal/mol for each ligand in the gas
phase. This stabilization, amounting to about 40 kcal/mol,
might be at the origin of the stability of such trianionic
complex.
(hydroxymethyl)-4H-pyran-4-one] and 2,2'-[(4-hydroxy-3-
methoxyphenyl)methanediyl]bis[3-hydroxy-6-hydroxymethyl)-
4H-pyran-4-one] ligands.52 Encouraged by these results, we
optimized the structures of the complexes Fe(HL)3 and [FeL3]3–
at the same level of theory as free ligand. In both cases, the
3d5 Fe3+ metal ion has been considered to assume a high-spin
configuration (spin sextet state). In the neutral complex, the
metal ion is chelated by the three HL– ligands in an only slightly
distorted octahedral fashion (O4-Fe-O13 angle = 96.15°; Fig. 7).
The three ligand units show corresponding bond lengths and
angles, with barely distinguishable Fe–O (“hydroxy”) and Fe–O
(“keto”) lengths in the complex (Fe-O4 and Fe-O5 distances =
2.082 and 1.994 Å, respectively, in the gas phase). A
comparison of the metric parameters optimized for Fe(HL)3 in
the gas phase (Fig. 7 and Table S8) with the corresponding
ones determined by X-ray diffraction for the neutral complexes
54
Fe(P2)3, isolated in Fe(P2)3·3H2O53, and Fe(P3)3 show a very
good agreement between the experimental and calculated
sets of data [P2 = 3-hydroxy-6-hydroxymethyl-1-methylpyrid-
4-onato; P3 = 1,2-dimethyl-3-hydroxypyrid-4-onato; average
structural Fe–O distances: 2.007 and 2.018 Å; C–O: 1.314 and
1.320 Å; O–Fe–O, 81.27° and 80.91 for Fe(P2)3 and Fe(P3)3,
respectively; average calculated Fe–O distances: 2.039 Å; C–O:
1.275 Å; O–Fe–O, 79.29° for Fe(HL)3.
Metal complexes
The complex formation equilibria of P1 with Al3+, Cu2+ and Zn2+
have been also studied by potentiometric methods and ESI-
MS. The estimated complex formation constants are reported
in Table 2 and the speciation plots in Figure 8.
1
Average metric parameters calculated from a search on the Cambridge Crystallographic Database (CSD 5.36 updated nov. 2014) on
entries with ref code: MOMLOR, BICSEL, CEZYIR, FUDRUR, FUDRUR10, GARWUR, GARWUR, JUTVOJ, JUTWAW, JUTWEA, KCATFE,
SUCCUO, SUCDAV, SUCDAV10, WEDDUF (CQ 1.17 build RC5).
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lipophilicity order was obtained from in silico VcieawlcArutilcaletOionnlinse,
using QikProp program.55 This program provides also a number
DOI: 10.1039/C6DT00129G
of indicators of the pharmacokinetic profile of a drug, as the
ability to cross the blood-brain barrier (log BB), the ability to
be absorbed through the intestinal tract to the bloodstream
(Caco-2 cell permeability) and the verification of Lipinski’s rule
of five 45 (Table 3).
Although P1 does not present any violation of the restricted
Lipinski´s rule, it presents some limitations to barrier-crossing
ability compared to DFP. In particular, P1 presents lower
permeability through the Caco-2 membranes (higher than 500
nm/sec is considered good)55, as well as through the blood-
brain barrier (log BB < -1.0, poorly distributed to the brain) and
thus lower bioavailability into the central nervous system.
Interestingly the tautomeric diphenolic form H2Lb is
characterized by slightly better pharmacokinetic descriptors
than H2La, namely in terms of membrane permeability, due to
the increased aromaticity of the molecule that involves a
lipophilicity enhancement.
Table 3. Experimental values of octanol/water coefficient log P for P1 (in the neutral
forms H2La and H2Lb) and DFP, and calculated values for clog P, ability to cross the
blood-brain barrier (log BB), ability to be absorbed through the intestinal track to the
blood (Caco-2 cell permeability), bioavailability into the central nervous system and
the verification of Lipinski’s rule of five.45
Name
MW
DFP
138
H2La
141
H2Lb
141
-
logPa
-0.82
0.915
-0.90
clogPb
-0.795 0.520
-1.021 0.978
log BBb,c
Caco-2 Permeability (nm/s)b
CNSb
-0.254
1141
+/-
192
--
0
256
-
0
Violations of Lipinsky rulesb
0
aoctanol/water partition coefficients, values measured in the present work
bPredicted values using program QikProp v.2.555
cblood/brain partition coefficient
Fig. 8. Speciation plots of P1 model complexes with A) Al3+, B) Cu2+ and C) Zn2+
metal ion concentration and 10 µM ligand concentration (log β in Table 2).
We want to remark that the pAl3+, pCu2+, pZn2+ calculated for
P1 complexes are almost equal to those for deferiprone and
3,4-hopo (see Table S32), in contrast with the net increase
relative to iron complexes. This behaviour drives us to define
P1 as an iron chelator more selective than deferiprone.
Lipophilicity and drug-like descriptors
Biodistribution studies
To evaluate the efficacy of the new ligand P1 as a chelating
agent for mobilization of metal ions, we studied the
biodistribution profile of the well-established radiotracer 67Ga-
citrate in female mice with simultaneous intraperitoneal
administration of the ligand solution. The effect of this
chelator on the 67Ga uptake and clearance on the major organs
and on the excretion can be viewed in comparison with the
drug DFP (Fig. 9).
Lipophilicity greatly influences the pharmacokinetic and
pharmacodynamic profile of
a drug, and the partition
coefficient between water and n-octanol (log P) is the
parameter in use for its evaluation. Log P of P1, and of DFP for
comparison, were measured with the “shake-flask” method.
The obtained values (Table 3) are quite similar, though that of
P1 (-0.90) is slightly less than that of DFP (-0.82). The same
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of pyridinone ligand into the catechol-like ligand; and (ii) the
View Article Online
DOI: 10.1039/C6DT00129G
log K 9.00 of P1, lower than that determined for deferiprone
by 0.82 units. The chelating ability of P1 toward aluminium,
copper and zinc ions is not higher than that of the
corresponding DFP and 3,4-hopo, and this renders P1 a more
selective iron chelator.
The bio-distribution studies on mice showed that P1 is as
effective as clinically-used DFP, notwithstanding the negative
charge on the complex existing at physiological pH (where the
species modelled as [FeL3H2]– is prevalent) could hinder in
some extent the crossing of membranes, and thus iron
excretion. Thus, suggesting that it is worthwhile to assess its in
vivo efficacy after oral administration. The bioavailability
parameters presented in Table 3 characterize P1 as a less
effective compound than DFP, even if the difference between
the parameters of H2La and H2Lb should mitigate this limit. The
study of metabolism of P1 in living organisms could be of
paramount importance, since it has been reported that 1’-
hydroxyalkyl substituents prevent glucuronidation in vivo in
hydroxypyridinones.
Fig. 9. Biodistribution data in the most relevant organs, expressed as % I. A./g for 67Ga-
citrate with simultaneous intraperitoneal injection of P1 or deferiprone, at 1, 24 and 48
h after administration in female Balb-C mice (n = 3).
Although the effect of DFP on the biodistribution of
radiotracer in metal-overload mice has been previously
reported, those studies involved a different animal model
(CD1)56,57 and so it was necessary to revaluate it herein for
Balb-C mice.
The co-administration of the ligand P1 enhanced the overall
excretion rate of radioactivity from whole animal body as
efficiently as DFP, 24 and 48 h after administration. No
significant differences in the radioactivity remaining in the
carcass and in the rate of radioactivity elimination were found,
except at 1 h.
The main differences in the distribution profile are related with
the blood clearance and the hepatic uptake at 1 h. P1 induces
slower clearance from blood as well as lower liver uptake than
DFP at 1 h after administration, which may be attributed to a
highest level of plasmatic protein binding, eventually due to
the 2-hydroxymethyl group and to some minor decrease on
the lipophilicity.
These overall promising results urge further studies to verify in
vivo if the toxicity, bioavailability and pharmacokinetic
properties of P1 can open a new way to future clinical trials.
Acknowledgements
GC and JIL acknowledge Regione Sardegna for the financial
support CRP-27564. VMN and MAZ acknowledge Regione
Sardegna for the financial support CRP-26712. DChL thanks the
support of the Intramural CSIC project 201530E011. MAS
acknowledges the Portuguese Foundation of Science and
Technology (FCT) for the financial support to the Project
UID/QUO/00100/2013 and Felloship to CQ.
Notes and references
Overall, this biodistribution profile, similar to that of DFP,
shows an increase of the radiotracer elimination rate and
attests the ability of the ligand to chelate the radiometal in
vivo.
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Conclusions
P1 combines the hydroxypyridinone and catechol features: at
low pH it coordinates efficiently metal ions as 5-hydroxy-2-
(hydroxymethyl)pyridin-4(1H)-one, while at basic pH as 2-
(hydroxymethyl)pyridine-3,4-diol.
The
presence
of
hydroxymethyl group favours the tautomeric equilibrium in
the free ligand, due to the formation of the hydrogen bond
N1…H8. The tautomeric transformation in the presence of
metal ions occurs at lower pH than in the free ligand. On the
basis of pFe3+ values, P1 should be considered a more efficient
iron chelator (pFe3+ 22.0) than the clinically-used DFP (20.67)49,
its 3,4-hopo analogues (20.77) and far more effective than
catechol (pFe 14.7).58 The high pFe3+ value of P1 depends on
two different factors, namely (i) the high stability of the iron
complexes, mainly attributed to the tautomeric rearrangement
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DOI: 10.1039/C6DT00129G
Complementary analytical techniques gave evidence that tautomeric changes of the new 5-
hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one positively influence its coordinating ability towards
Fe3+.