Synthesis, chemical and biological studies on new Fe3+-glycosilated β-diketo complexes for the treatment of iron deficiency

Article abstract of DOI:10.1016/j.ejmech.2008.02.045

A simple synthetic pathway to obtain glycosilated β-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore we may suggest the use of their Fe³⁺-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH₃) are characterized by means of spectroscopic (UV, ¹H and ¹³C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t_1/2 > 100 h) and show a low cytotoxicity also in high concentrations (IC₅₀ > 400 μM). They are able to bind Fe³⁺ ion in acid condition (pH ～ 2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (log P > -0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca²⁺, and in particular 6-GlcOCH₃ is able to inhibit lipid peroxidation.

Full text of DOI:10.1016/j.ejmech.2008.02.045

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2549e2556
http://www.elsevier.com/locate/ejmech
Original article
3þ
Synthesis, chemical and biological studies on new Fe -glycosilated
b-diketo complexes for the treatment of iron deficiency
a
a
a
c
b
Beatrice Arezzini , Marco Ferrali , Erika Ferrari , Chiara Frassineti , Sandra Lazzari ,
a
b
b,
Gaetano Marverti , Ferdinando Spagnolo , Monica Saladini *
a
c
Department of Physiopathology and Experimental Medicine, University of Siena, Via Aldo Moro 53100 Siena, Italy
b
Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy
Department of Biomedical Science, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy
Received 4 October 2007; received in revised form 25 February 2008; accepted 29 February 2008
Available online 14 March 2008
Abstract
A simple synthetic pathway to obtain glycosilated b-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore
3þ
we may suggest the use of their Fe -complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-
1
13
3
) are characterized by means of spectroscopic (UV, H and C NMR) and potentiometric techniques; they have a good
GlcOH and 6-GlcOCH
water solubility, are kinetically stable in physiological condition (t1/2 > 100 h) and show a low cytotoxicity also in high concentrations
3
þ
(
IC50 > 400 mM). They are able to bind Fe ion in acid condition (pH w 2) forming complex species thermodynamically more stable than those
of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and
physiological pH and have a good lypophilicity (log P > ꢀ0.7) that suggests an efficient gastrointestinal absorption in view of their possible use
2þ
in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca , and in particular 6-GlcOCH is
3
able to inhibit lipid peroxidation.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Iron complexes; b-Diketo ligands; Curcumin; Kinetic study; Thermodynamic study; Cytotoxicity
1
. Introduction
constipation, resulting in poor compliance [4,5]. In case of inad-
equate gastrointestinal iron absorption or intolerance to the req-
uisite dose of oral iron a parenteral therapy is preferred [6].
Parenteral iron formulations containing ironesucrose com-
plexes are widely used (Venofer , Ferrlecit ). A parenteral
iron preparation must have a complex ability sufficient to avoid
excessive blood concentration of Fe and low toxicity to avoid
side effects [7]. Parenteral iron administration is mainly used
in treatment of anaemia, in chronic haemodialysis patients
and in serious iron deficiency such as in the last month of preg-
nancy. Great interest is turned on the development of new safe
and low toxic iron carriers both for oral and intravenous
administration.
Iron plays an important role in the living systems, therefore
iron deficiency causes anaemia and other pathological changes
in the body. In order to prevent and treat this disease some iron
containing medicines are used. The advantages and disadvan-
tages of the various preparates are related to the root of admin-
istration [1e3]. Under normal circumstances orally
administrated iron is the treatment of choice since it is simple,
effective, safe and cheap. Actually different iron supplements
Ò
Ò
2
þ
3þ
Ò
containing Fe or Fe complexes or salts (Iron Complex ,
Ò
Ferro-grad ) are in use. However, oral Fe administration causes
various malaises such as nausea, vomiting, pain, diarrhoea and
Previously we have explored the ability of natural occurring
molecules such as curcumin (Scheme 1) and its derivatives to
3
chelate Fe ion [8]; the b-diketo moiety of curcumin demon-
strated coordination ability toward this metal ion. Its main
þ
*
Corresponding author. Tel.: þ39 0592055040; fax: þ39 059373543.
E-mail address: monica.saladini@unimore.it (M. Saladini).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.045

2550
B. Arezzini et al. / European Journal of Medicinal Chemistry 43 (2008) 2549e2556
HO
O
2. Experimental section
2
2
.1. Synthesis
OH
HO
.1.1. 2,3,4,6-Tetracetyl-a-D-glucopyranosyl bromide (1)
Compound 1 was synthesized quantitatively by reaction of
1,2,3,4,6-pentaacetyl-b-D-glucopyranose with HBr 30% in
OCH3
OCH3
Scheme 1. Curcumin.
glacial acetic acid, at room temperature for 4 h [16].
drawback in the clinical use is its low water solubility; to over-
come this handicap we have synthesized glycosylated curcu-
minoids [9]. These compounds add iron chelating ability to
good water solubility and free-radical scavenging activity, in-
hibiting the iron redox cycle. In order to reduce molecular
weight and favour diffusion through cell membranes, we
have also synthesized new glycolsilated compounds derived
from curcumin (Scheme 2). For one of them (6-GlcOCH3),
a preliminary study on its iron chelating properties has been
previously explored [10]. Before proposing these complexes
for clinical use as iron supplements in oral therapy, a complete
characterization of complexes and ligands have to be done to
assure their safety.
2.1.2. 4-[(2,3,4,6-Tetracetyl-b-D-glucopyranoside)
oxy]benzaldehyde (2); 3-hydroxy-4-[(2,3,4,6-tetracetyl-b-D-
glucopyranoside)oxy]benzaldehyde (3); 3-methoxy-
4-[(2,3,4,6-tetracetyl-b-D-glucopyranoside)oxy]-
benzaldehyde (4)
The appropriate benzaldehyde was solubilized in NaOH, and
then slowly dropped in an equimolar solution of 1 in acetone.
The reaction was kept under stirring overnight at room temper-
ature, and a precipitate splitted up when water was added. The
isolated product was recrystallized from EtOH.
2.1.3. 4-Hydroxy-6-{4-[(2,3,4,6-tetracetyl-
b-D-glucopyranoside)oxy]phenyl}hex-3,5-dien-2-one (5);
4-hydroxy-6-{3-hydroxy-4-[(2,3,4,6-tetracetyl-b-D-
glucopyranoside)oxy]phenyl}hex-3,5-dien-2-one (6);
4-hydroxy-6-{3-methoxy-4-[(2,3,4,6-tetracetyl-b-D-
In this paper we report a new simplified synthetic pathway
with enhanced yield and reduced reaction time for compounds
showed in Scheme 2. The effect of the aromatic ring substitu-
ents on iron complexes’ stability is evaluated together with the
glucopyranoside)oxy]phenyl}hex-3,5-dien-2-one (7)
2
þ
ꢀ3
affinity toward other physiological metal ions such as Ca
A
suspension of 4.0 ꢁ 10 mol of B O
and
2
3
ꢀ3
which may compete with iron in the body. Lipophilicity is
an important factor that influences drug absorption by gastro-
intestinal tract, anyway the chemical stability of the metal car-
rier in physiological conditions is not less important; so both
these aspects are investigated here.
In order to evaluate a possible cytotoxicity of these com-
pounds against normal tissues as a prediction of side effects,
we have tested the molecules on the growth of Vero cells.
The Vero cell line, an immortal, non-tumorigenic fibroblastic
cell line established from kidney cells of the African green
monkey (Cercopithecus aethiops), has a characteristic growth
pattern in culture [11], and has been used to study cell growth,
differentiation [12,13], and cytotoxicity [14]. These cells are
recommended for such investigations in standard protocols
4.0 ꢁ 10 mol of acetylacetone in DMF (2.0 mL) was stirred
ꢂ
for 10 min at 80 C, followed by the addition of
ꢀ
3
16.0 ꢁ 10 mol of tributylborate. After 10 min a solution of
ꢀ
3
0.8 ꢁ 10 mol of the appropriate glucosilbenzaldehyde (2e4)
ꢀ3
in 1.5 mL of DMF and a solution of 0.8 ꢁ 10 mol of n-
butylamine (ButNH ) in 1.5 mL of DMF were slowly dropped
2
ꢂ
(1 h). The solution, kept under stirring at 80 C for 4 h, devel-
oped a yellow-orange colour. The solution was emptied into
15 mL of hot (60 C) 5% aqueous acetic acid. During the acid-
ꢂ
ification, the solution was cooled to room temperature. The
crude product appeared as heavy oil. After about 1 h of stir-
ring, the solvent was removed and the syrup suspended in
water at room temperature, as stirring continued, slowly turned
into a solid. The crude solid was collected and purified with
flash chromatography (silicagel 0.035e0.070 mm; EtOAc/
[15] and provide an excellent model for studying alterations
in cell growth induced by chemicals in vitro because of their
characteristic growth pattern and behaviour in culture.
EtPet). Elemental analysis: 5 e C26H O12 calcd: C 57.99,
34
H 6.36; experimental: C 58.15, H 6.48; yield 88%; 6 e
C H O Calcd: C 56.31, H 6.18; experimental: C 56.15,
2
6 34 13
H 6.50; yield 63%; 7 e C H O calcd: C 57.04, H 6.38;
2
7 36 13
experimental: C 57.60, H 6.45; Yield 42%; NMR chemical
shifts (ppm in CDCl ): 5 e H-1 2.16, H-3 5.63, H-5 6.36,
H-6 7.50, H-8 7.43, H-9 7.09, H-11 7.09, H-12 7.43; C-1
26.7, C-2 197.4, C-3 100.6, C-4 177.1, C-5 121.6, C-6
HO
4
O
2
3
OH
8
17
1
9
2
6
1
11
7
8
5
3
1
1
6
O
HO
HO
10
138.6, C-7 130.3, C-8 129.2, C-9 116.9, C-10 157.9, C-11
116.9, C-12 129.2; 6 e H-1 2.16, H-3 5.63, H-5 6.34, H-6
7.48, H-8 7.14, H-11 6.96, H-12 7.02; C-1 27.0, C-2 197.9,
14
O
15
13
OH
R
R = H
6-GlcH
6-GlcOH
OCH3 6-GlcOCH3
C-3 101.1, C-4 176.7, C-5 122.5, C-6 138.8, C-7 132.3, C-8
14.6, C-9 147.3, C-10 145.3, C-11 117.1, C-12 120.1; 7 e
OH
1
H-1 2.17, H-3 5.64, H-5 6.36, H-6 7.52, H-8 7.08, H-11
7.04, H-12 7.07; C-1 26.9, C-2 197.7, C-3 101.0, C-4 177.0,
Scheme 2. Synthesised compounds.

B. Arezzini et al. / European Journal of Medicinal Chemistry 43 (2008) 2549e2556
2551
2
þ
þ
C-5 122.2, C-6 139.1, C-7 131.6, C-8 119.7, C-9 150.9, C-10
1
[19] taking into account [Fe(OH)] and [Fe(OH)2] species
[20]. The protonation constants of the ligands were determined
47.6, C-11 121.3, C-12 111.6, CeR 56.1.
ꢀ3
3þ
2þ
by titration of 3 ꢁ 10 M solutions. The Fe /ligand and Ca
/
ligand systems were investigated in 1:1, 1:2 and 1:3 molar ratios
2
.1.4. 4-Hydroxy-6-{4-[(b-D-glucopyranoside)
3
þ
2þ
ꢀ3
ꢀ3
oxy]phenyl}hex-3,5-dien-2-one (6-GlcH) (8); 4-hydroxy-
([Fe ] and [Ca ] ranges 3 ꢁ 10 e1 ꢁ 10 M). Aqueous
ꢀ
2
6
5
-{3-hydroxy-4-[b-D-glucopyranoside)oxy]phenyl}hex-3,
-dien-2-one (6-GlcOH) (9); 4-hydroxy-6-{3-methoxy-4-
NaOH (1 ꢁ 10 M) was used as titrant. A measuring time of
600e720 s for each point was used. At least 10 measurements
were performed for each system with 40 data points in each ti-
tration in the pH range 2e10.
[
(b-D-glucopyranoside)oxy]phenyl}hex-3,5-dien-2-one
(
6-GlcOCH ) (10)
3
The acetylated compounds, solubilized in methanolic 0.1 M
solution of CH ONa, were kept under stirring for 2 h; pH was
2.4. Partition coefficient
3
brought to neutrality by use of a DOWEX ion exchange resin,
which was finally filtered off, and solutions were evaporated
under vacuum. The reaction is quantitative. Elemental analy-
sis: 8 e C H O calcd: C 59.01, H 6.05; experimental: C
The shake-flask method was performed on each ligand and
on the M/L 1:3 system at pH 7.0 (NaH PO /Na HPO ). With
2
4
2
4
a known volume of water (15e30 mL) previously saturated
with high purity analytical grade n-octanol, 25 mL of the
1
8 22 8
5
mental: C 56.40, H 5.40; 10 e C H O calcd: C 57.57, H
8.96, H 5.89; 9 e C H O calcd: C 56.54, H 5.80; experi-
18 22 9
ꢀ
4
ligand stock solution (10 M) or M/L 1:3 solution (10 M)
ꢀ4
1
9 24 9
ꢂ
6
.10; experimental: C 57.43, H 6.37.
were mixed in a vessel and then shaken at 37 C for 4 h. After
separation of the two phases, the ligand concentration in aque-
ous solution was determined by spectrophotometric method
using a Jasco V-570 spectrophotometer.
2
.2. Spectroscopy
Spectrophotometric titrations were performed using Jasco V-
ꢂ
5
70 spectrophotometer at 25.0 ꢃ 0.1 C in the 200e600 nm
2.5. Cytotoxicity assays
ꢀ5
spectral range employing a 1 cm quartz cell. 2.5 ꢁ 10
M
aqueous solutions of ligands were investigated varying the pH
value by adding small amounts of concentrated NaOH or HCl
in the pH range 1e11. The protonation constants of the ligands
were evaluated from spectrophotometric titrations using the
pHab2000 software [17]. Constant additions (10 mL each
Vero cells, obtained from the ‘‘Istituto Zooprofilattico’’
(Brescia, Italy), were maintained in MEM medium (Whittaker
Bioproducts, Walkersville, MD, USA) supplemented with
10% heat-inactivated fetal calf serum (Gibco/BRL, Gaithers-
burg, MD, USA) and penicillin G (100 mg/mL), streptomycin
(100 mg/mL) (Sigma Chemical Company, MO, USA). Cul-
3
þ
ꢀ2
time) of Fe solution (10 M) were performed using a micro-
pipette in order to reach different M/L molar ratios.
tures were equilibrated with humidified 5% CO in air at
2
ꢂ
The chemical stability of the ligands was evaluated as
a change in absorbance at 345 nm. The ligands solutions of
37 C. The medium was renewed at 48 h intervals, and the
cells were always subcultured when the monolayers become
confluent.
The cytotoxicity of compounds 6-GlcH, 6-GlcOH, 6-
ꢀ
5
ꢀ2
5
lution (pH 7). The ligands stability was investigated also in
ꢁ 10 M were prepared in 10 M phosphate-buffered so-
3
þ
in M/L 1:1 molar ratio at pH 2 (HCl
presence of Fe
.01 M) and pH 7 (NaH PO /Na HPO ). A constant ionic
strength of 0.1 M (NaNO ) was maintained in all experiments.
GlcOCH and Curcumin on Vero cells was assessed by a modi-
3
0
fication of the crystal violet dye assay [21]. In this protocol,
4
2
4
2
4
Vero cells were seeded at a density of 4 ꢁ 10 cells/well of
3
ꢂ
The cell was stored at 37 C in the dark.
NMR spectra were recorded on a Bruker Avance AMX-400
spectrometer with a Broad Band 5 mm probe (inverse detec-
a 24-well culture plate (Sarstedt, Numbrecht, Germany) and
ꢂ
cultured for 72 h at 37 C (control cells). Treated cells were in-
cubated with different concentrations of the compounds and
were grown under the same conditions as non-treated (control)
cells. After incubation, the culture medium was removed and
the cells were washed with 0.1 mL of 0.1 M phosphate-buffered
1
3
tion). Nominal frequencies are 100.13 MHz for C and
1
00.13 MHz for H. The typical acquisition parameters for
4
1
H are as follows: 20 ppm spectral bandwidth (SW), 6.1 ms
1
ꢂ
ꢂ
pulse width (90 pulse hard pulse on H), 0.5e1 s pulse delay,
16e512 number of scans. For each ligand 0.5 mL of
saline (PBS), pH 7.4 at 37 C, then cell monolayer was fixed
2
a 10 M solution was prepared in CD OD, then 10 additions
with methanol prior to staining with 0.25% crystal violet solu-
tion in 80% absolute ethanol for at least 30 min. After washing
several times with distilled water to remove the excess dye, the
cells were let to dry. The incorporated dye was solubilized in
acidic isopropanol (1 N HCl/2-propanol, 1:10). After appropri-
ate dilution, dye was determined spectrophotometrically at
ꢀ2
3
ꢀ
2
(
in CD OD) were performed using a micropipette.
10 mL each time) of Ga(NO ) or CaCl solution (5 ꢁ 10
M
3
3
2
3
2
.3. Potentiometry
540 nm. The extracted dye was proportional to cell number. Per-
Potentiometric measurements were performed in aqueous
ꢂ
centage of survival was calculated by comparing the absor-
bances of exposed to that of non-exposed (control) cultures.
Rat liver microsomes were prepared in Trisemaleate buffer
(pH 7.4) following a standard procedure and resuspended in
the same buffer at the concentration of 2.5 mg protein/mL
solution at 25.0 ꢃ 0.1 C according to general procedures previ-
ously reported [18]. A constant ionic strength of 0.1 M (solid
NaNO ) and nitrogen gas was maintained in all experiments.
The stability constants were refined with Hyperquad program
3

2552
B. Arezzini et al. / European Journal of Medicinal Chemistry 43 (2008) 2549e2556
Table 1
1
H chemical shifts (d ppm) of ligands, their gallium(III) complexes and Dd (dcomplex ꢀ dligand) registered at 300 K in CD
3
OD
H-1
H-3
H-5
H-6
H-8
H-9
H-11
H-12
HeR
6
-GlcH
2.17
2.21 (0.04)
2.25 (0.08)
5.82
5.91 (0.09)
6.57
6.72 (0.15)
6.76 (0.19)
7.58
7.79 (0.21)
7.86 (0.28)
7.58
7.63 (0.05)
7.65 (0.07)
7.15
7.16 (0.01)
7.17 (0.02)
7.15
7.16 (0.01)
7.17 (0.02)
7.58
7.63 (0.05)
7.65 (0.07)
e
e
e
M/L 1:3
M/L 1:1
a
6
-GlcOH
2.17
2.21 (0.04)
2.25 (0.08)
5.81
a
6.51
6.67 (0.16)
6.70 (0.19)
7.51
7.72 (0.21)
7.79 (0.28)
7.14
7.18 (0.04)
7.20 (0.06)
7.23
7.25 (0.02)
7.26 (0.03)
7.07
7.11 (0.04)
7.14 (0.07)
e
e
e
M/L 1:3
M/L 1:1
a
6
-GlcOCH
3
2.10
2.19 (0.09)
2.24 (0.14)
5.83
5.90 (0.07)
6.00 (0.17)
6.59
6.73 (0.14)
6.77 (0.18)
7.57
7.75 (0.18)
7.82 (0.25)
7.20
7.30 (0.10)
7.31 (0.11)
7.20
7.20 (0.00)
7.21 (0.01)
7.29
7.20 (0.09)
7.21 (0.08)
3.94
3.92 (0.02)
3.92 (0.02)
M/L 1:3
M/L 1:1
a
The methylenic proton is mobile, in fast exchange with residual HOD.
ꢂ
2þ
[9]. Microsomal membranes were incubated at 37 C at the
concentration of 1.25 mg/mL of protein in the presence of
1
and 2). By adding Ca to the ligand solution no resonances’s
shifts are observed, even in high excess of metal ions and in-
3
00 mM Fe e2.5 mM ADP and different concentrations of
þ
2þ
creasing pH, suggesting a poor ability to bind Ca ion.
the complexants (100e300 mM) dissolved in small volumes
of ethanol. Samples of the mixture were withdrawn at the
indicated times and the lipid peroxidation measured with
a standard procedure [9].
3.2. UVevis spectroscopy
All the compounds are characterised by a maximum of ab-
sorbance in the 320e345 nm spectral range with a red shift on
increasing pH (340e365 nm). By plotting absorbance vs. pH,
one equivalent point is observed corresponding to enolic pro-
ton dissociation for all the compounds, while for 6-GlcOH
a second equivalent point corresponding to phenolic proton
3
. Results and discussion
3
.1. NMR spectroscopy
Ligand molecules show a typical spectrum of a b-keto-eno-
dissociation is found; the pK
photometric data are reported in Table 3.
values calculated from spectro-
a
lic moiety [9,22], the enolic proton is mobile, in fast exchange
with residual HOD and implied in a strong intra-molecular hy-
drogen bond, as confirmed by crystal structure of analogous
compounds [23], that makes it equally shared by the two ox-
ygen atoms. NMR signal of enolic proton is a very broad
peak around 16e17 ppm, depending on the substituent.
3
þ
By adding Fe solution to the free ligands in acidic condi-
tion (pH w 4.5) a general increase in absorbance is observed
and a new band appears around 400 nm (Fig. 1), due to the in-
3
þ
teraction of Fe with the b-diketo moiety with an anticipated
enolic dissociation with respect to the free ligand (DpK w 4).
a
3
þ
Ga(NO ) is used as NMR probe instead of Fe ; its addi-
3
The absorbance rises with M/L molar ratio and reaches a max-
imum at M/L 1:1 as detected by NMR data.
3
tion to the ligand solution, at acidic pH, immediately origi-
nates a complex species, in slow chemical exchange in
NMR time scale, whose spectral pattern resembles one of
the free ligands but is strongly shifted downfield. The com-
plete formation of this species is reached when M/L is 1:3,
with disappearance of the signals of the free ligand. As long
as gallium ion is added a new downfielded species appears,
hinting the formation of another complex species, character-
ized by a M/L 1:1 independently on the ligand (Tables 1
3.3. Kinetic study
For all the ligands and their Fe/L 1:1 systems, the plotting
of ln(A /A ) versus time gives a linear regression, hinting a first
t
0
ꢂ
order kinetic process at 37 C and constant ionic strength.
Fig. 2 shows that ligands’ degradation process is extremely
slow being the percentage of decomposed ligand less than
Table 2
1
3
3
C chemical shifts (d ppm) of ligands and their gallium(III) complexes registered at 300 K in CD OD
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
CeR
6
-GlcH
27.3
28.4
28.5
200.1
197.6
198.0
102.5
103.4
103.9
179.3
186.1
186.4
123.0
126.1
125.6
141.4
144.1
145.3
131.6
131.3
130.9
131.4
131.9
132.0
118.9
119.0
118.9
163.1
161.8
162.0
118.9
119.0
118.9
131.4
131.9
132.0
M/L 1:3
M/L 1:1
6
-GlcOH
27.8
28.1
28.4
199.4
197.8
198.6
102.6
103.7
104.2
178.4
186.0
186.6
123.3
126.1
125.7
141.6
144.0
145.8
132.9
132.5
132.3
116.7
116.7
117.2
149.4
149.6
149.6
149.5
149.6
149.6
119.2
118.9
119.0
122.8
123.0
123.7
M/L 1:3
M/L 1:1
6
-GlcOCH
3
27.5
28.2
28.4
200.0
197.4
198.3
102.7
103.6
104.1
178.9
186.1
186.5
123.1
126.2
125.2
141.1
143.9
145.4
132.2
131.9
131.4
118.3
118.1
117.8
151.7
151.9
151.8
150.4
150.9
151.3
123.9
124.5
124.8
113.0
113.2
113.4
57.5
57.5
57.5
M/L 1:3
M/L 1:1

B. Arezzini et al. / European Journal of Medicinal Chemistry 43 (2008) 2549e2556
2553
Table 3
Logarithms of protonation constants of the ligands calculated using spectrophotometric and potentiometric data
a
6-GlcH
6-GlcOH
6-GlcOCH
3
b
b
b
pKa1
pKa2
9.26(3) 9.22(5)
8.03(6) 8.03(9)
9.44(7) 9.48(6)
8.75(2) 8.73(2)
b
3
þ
3þ
2þ
3þ
3þ
2þ
3þc
3þ
2þ
Fe
10.22(8)
6.77(5)
Ga
10.39(7)
7.04(5)
Ca
2.69(6)
Fe
9.5(1)
4.48(9)
Ga
Ca
1.56(7)
Fe
9.71(3)
5.83(4)
Ga
Ca
2.11(5)
[
[
ML]
ML(OH)]
9.1(1)
4.98(9)
9.6(2)
6.01(7)
[
ML
ML
2
]
(OH)]
18.61(5)
12.37(7)
19.23(7)
14.77(9)
5.56(9)
18.2(1)
13.2(1)
17.8 (1)
13.1(1)
5.72(5)
18.7(2)
14.2(4)
19.02(5)
13.5(5)
5.34(4)
[
2
[ML
3
]
25.59(8)
27.05(9)
26.0(2)
25.7(2)
27.3(8)
26.8(7)
ꢂ
Formation constants of complex species are estimated using potentiometric data [T ¼ 25 C, I ¼ 0.1 M (NaNO
3
)].
a
For 6-GlcOH log K values are evaluated taking into account the overall stability constants and protonation constants of the ligand.
Spectrophotometric data.
Values taken from Ref. [10].
b
c
1
0% after 8 h at pH 7, while curcumin, in similar conditions, is
effect of the substituent in meta position lowers the pK value
a
almost completely decomposed after 1 h [24]. The presence of
sugar moiety and the reduced conjugation in molecular skele-
ton of our ligands with respect to curcumin, delays degrada-
tion processes by increasing t1/2 (>100 h for all our
compounds vs. 10 min of curcumin) resembling the stability
of cyclodextrin complexed curcumin (t1/2 > 100) [25]. The ki-
netic stability of the ligands is also confirmed by NMR data, in
in the order 6-GlcOH < 6-GlcOCH < 6-GlcH.
3
3
þ
The species distribution curves of Fe complexes of 6-
GlcH are reported in Fig. 3, while Table 3 shows logarithms
of calculated stability constants. At acidic pH (w2) the pre-
vailing species is [FeL], confirming UV and NMR data. In
3
þ
this complex Fe is coordinated by the ligand molecule act-
ing as chelating agent through the dissociated keto-enolic moi-
ety. On increasing pH the complex system may suffer from the
1
fact no relevant changes in H chemical shifts and integrated
ꢀ
areas are observed in the spectra during the first 72 h. The na-
ture of the substituent doesn’t drive the degradation process,
although 6-GlcH is the more stable one.
competition of OH groups till Fe(OH) precipitation, as re-
3
ported in the distribution curves obtained taking into account
Kps of Fe(OH) [26]; anyway, once iron is uptaken from the
3
3
þ
At physiological pH the presence of Fe in M/L 1:1 molar
ratio seems not to influence the decomposition process, while
the same system in acidic condition (pH ¼ 2) revealed a higher
stability (degradation after 8 h < 5%).
gastrointestinal tract, Fe(OH) precipitation is prevented by
3
the interaction with specific Fe-proteins deputated to iron
homeostasis in biological systems.
3
þ
A comparison between Fe -complexes with our ligands and
those with gluconic acid [27], which is the most widely used
3
þ
Fe -supplier in treatment of anaemia, is forbidden on the basis
of stability constant values due to the different stoichiometry of
the formed species. Anyway the amount of free iron at acidic pH
3
.4. Potentiometric titrations
The protonation constants of the ligands were determined
(w2) is around 60% for gluconic acid, 20% for 6-GlcOH, 30%
both by spectrophotometric and potentiometric methods and
the differences range from 0.02 to 0.04 (Table 3). Potentiomet-
ric analysis gives more accurate results than other techniques;
therefore, we decided to evaluate log K of the complexes only
by potentiometry. The substituent on the aromatic ring influ-
1
00
98
96
94
ences the pK value of the ligands; the electron-withdrawing
a
1
0
0
0
0
.8
.6
.4
.2
2
.5
1
1
0
9
9
2
0
6-GlcH
0.00
0.20
0.40
0.60
0.80
1.00
6
6
-GlcOH
M/L
-GlcOCH3
.5
0
0
2
4
6
8
Time (h)
2
40
290
340
390
nm
440
490
540
Fig. 2. Plotting of residual compound percentage versus time (h) for all ligands
(
continuous line) and Fe/L 1:1 systems (broken line). Spectrophotometric
ꢂ
Fig. 1. Spectrophotometric titration of 6-GlcOH solution with Fe(NO
3
)
3
. The
measurements were performed in buffer solution (pH ¼ 7) at 37 C in the
ꢀ
5
inset shows the plot of absorbance vs. M/L molar ratio at l ¼ 400 nm.
dark, [L]
0
¼ 2.5 ꢁ 10 M.

2554
B. Arezzini et al. / European Journal of Medicinal Chemistry 43 (2008) 2549e2556
100
Fe(OH)3(s)
A
B
C
Fe(OH)3(s)
80
60
40
20
0
FeL
Fe(OH)3(s)
FeL3
FeL
FeL2
FeL
Fe
FeL3
Fe
FeL(OH)
FeL2
4
Fe
FeL2
4
2
6
8
2
4
6
8
2
6
8
pH
3
Fig. 3. Species distribution curves of Fe /L systems: (A) Fe/6-GlcH; (B) Fe/6-GlcOH and (C) Fe/6-GlcOCH ; [Fe ]
þ
3þ
total ¼ 1 ꢁ 10ꢀ3 M, [L]total ¼ 2 ꢁ 10ꢀ3 M.
3
for 6-GlcH and 40% for 6-GlcOCH suggesting a higher stabil-
3
ability is described by the partition coefficient between n-oc-
tanol and buffered aqueous solution (pH ¼ 7.0). Table 4 re-
ports the log P values for both ligands and complexes. The
log P value of the ligands is mostly influenced by the nature
of the substituent in meta position in the aromatic ring: the
ity of our complexes with respect to gluconic acid; this feature is
fundamental for a good gastrointestinal absorption in view of
a potential oral administration.
2
þ
As Ca is one of the most abundant metal ion in human
body, being its concentration in physiological fluids about
increased hydrophilic character of OCH with respect to H
3
1
iron in the interaction with our ligands. Fig. 4 shows the distri-
bution curves of Ca /6-GlcOH system with respect to the
ligand, calculated using the stability constants reported in Table
.2 mM [28], we have evaluated its possible competition with
decreases the log P value, while the presence of OH favours
the formation of intra-molecular hydrogen bond interactions
with the glycosidic moiety diminishing the hydrophilicity of
the compound [29]. The lipid solubility of Fe-complexes is
lower than that of the corresponding ligand but is sufficient
to give an efficient absorption from the gastrointestinal tract
(log P > ꢀ0,7) [30]. A linear relationship is often used to cor-
relate the partition coefficients of the free ligand (L) and iron
complex (FeL) [31]:
2þ
3
. Up to physiological pH, the affinity of 6-GlcOH for proton is
higher than that for calcium, whereas in basic condition
pH w 9) only 20% of the ligand is coordinated to calcium
(
ion. This percentage dramatically decreases in presence of
iron (Fe/Ca 1:5) confirming the poor affinity for calcium ion
1
as already observed by H NMR data.
log PFeL ¼ n log P þ k
L
3
.5. Partition coefficients
where n is the stoichiometry of the complex and k is a constant
representing the change in hydrophobicity in converting the li-
gating group of the ligand into the iron coordination sphere of
the complex. Our data match this behaviour with a calculated
n ¼ 0.95 and k ¼ ꢀ0.11.
The efficient penetration through biomembranes is a pre-
requisite for oral administration of Fe containing drugs; this
100
3.6. Cytotoxicity
LH
8
6
4
2
0
0
0
0
0
In order to evaluate the possible drawbacks of the free li-
gands, upon iron delivery, we have tested the inhibitory effect
in vitro of the compounds against the growth of Vero cells, cho-
sen as control cell line. 6-GlcOH treatment decreases Vero cell
proliferation by nearly 10% at 400 mM and by 20% at 800 mM
LH-1
L
Table 4
Logarithms of partition coefficient defined as the concentration ratio (mol L
ꢀ1
CaL
CaL2
)
in the two solvents (n-octanol and water)
Ligand
Feeligand
system
2
4
6
8
10
pH
6
-GlcH
6-GlcOH
0.39
0.32
0.13
0.28
0.17
0.02
2þ
Fig. 4. Species distribution curves for Ca /6-GlcOH system;
2
þ
ꢀ3
ꢀ4
6-GlcOCH
3
[Ca
]
total ¼ 1.2 ꢁ 10 M, [L]total ¼ 7.5 ꢁ 10 M.

B. Arezzini et al. / European Journal of Medicinal Chemistry 43 (2008) 2549e2556
2555
VERO cells
8
7
6
5
4
3
2
1
0
c
A
B
C
d
1
00
*
b, e
7
5
2
5
0
5
0
*
*
a
*
0'
10'
20'
30'
60'
1
1
4
2
d
*
e
10
8
c
6
6
6
-GlcH
-GlcOH
-GlcOCH3
6
b
4
*
*
0
200
400
600
800
2
[
drugs] µM
a
0
0'
10'
20'
30'
60'
Fig. 5. Survival of VERO cells following treatment with 6-GlcH (A), 6-
GlcOH (B) and 6-GlcOCH (C). Each point represents mean value ꢃ SD of
3
7
6
5
4
3
2
1
0
3
separate experiments, each conducted with triplicate plates.
b
c
(
Fig. 5). This inhibition represents an 80-fold lower cytotoxic
potency of the compound when compared to curcumin, which
shows to inhibit cell growth to the same extent but at greater
concentrations (5 and 10 mM for 10% and 20% inibition, re-
spectively). The substitution of OH group with OCH group re-
3
sults in a higher cytotoxicity that is dose-related, since at
00 mM the growth of Vero cells is inhibited by 40% and by
5% at 800 mM, showing an IC50 value of nearly 700 mM in
the tested drug concentration range. 6-GlcH decreases Vero
cell growth by approximately 2% (600 mM) and 40%
d
e
*
6
6
*
a
*
*
10'
0'
20'
30'
60'
Fig. 6. Time course of thiobarbituric acid reacting substances (TBARS) forma-
3þ
ꢂ
(
800 mM). These results indicate that the three compounds
tion in liver microsomes (MC) incubating at 37 C in presence of Fe and:
(A) 6-GlcH; (B) 6-GlcOH; (C) 6-GlcOCH ; (a) control MC; (b)
might be well tolerated by normal tissues up to 400 mM, in par-
ticular, 6-GlcH seems to be non-toxic up to 600 mM and 6-
GlcOH even up to 800 mM. Despite their different behaviour
all the compounds show a cytotoxicity extremely lower than
curcumin, which is commonly administrated even in high dos-
ages without relevant side effects [32].
Furthermore in vitro experiments to evaluate the effects of
iron complexes on subcellular fractions were performed. Rat
liver microsomes were incubated with the three compounds,
3
3
þ
MC plus 100 mM Fe e2.5 mM ADP; (c)
d) as (b) plus 200 mM chelator; (e)
The values represent the mean of two experiments.
as (b) plus 100 mM chelator;
as (b) plus 300 mM chelator.
(
*
or 6-GlcH, is suitable for iron administration as 1:3 complex.
Despite the obvious limitations of the in vitro observations,
these results indicate that 6-GlcOCH maintains iron in a redox
3
inactive form before its delivery. On the other hand, its cytotox-
icity appears to be relatively low, so that its possible use as iron
donor can be worthy of attention.
3
þ
each in the presence of three different Fe ratios (Fig. 6). As
it can be seen, the presence of 6-GlcH (Fig. 6A) or 6-GlcOH
(
Fig. 6B), does not attenuate the extent of lipid peroxidation in-
3
þ
duced by 100 mM Fe . When 6-GlcOH is added (Fig. 6B), iron
redox activity is even stimulated, indicating a weak bond be-
tween iron and the compound which results in a greater redox
Acknowledgments
We are grateful to the ‘‘Centro Interdipartimentale Grandi
Strumenti e C.I.G.S.’’ of the University of Modena and Re-
ggio Emilia and to the ‘‘Fondazione Cassa di Risparmio di
Modena’’ for supplying NMR spectrometer.
capability of iron. When 6-GlcOCH (Fig. 6C) is present at
3
the 1:3 (Fe:L) ratio, the lipid peroxidation is inhibited within
3
0 min suggesting that this compound, rather than 6-GlcOH

2
556
B. Arezzini et al. / European Journal of Medicinal Chemistry 43 (2008) 2549e2556
[14] P. Estacia, A.R. Santos Jr., P.L. Moreira, S.C. Genari, Braz. J. Morphol.
Sci. 19 (2002) 41.
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[
(