Bis-maltol-polyamine family: structural modifications at strategic positions. Synthesis, coordination and antineoplastic activity of two new ligands

Article abstract of DOI:10.1039/d0nj05327a

Two new maltol-based ligands are presented,L1(N,N′-bis((3-hydroxy-6-methyl-4-pyron-2-yl)methyl)-N,N′-dimethylethylenediamine) andL2(N,N′-bis((3-hydroxy-6-hydroxymethyl-4-pyron-2-yl)methyl)-N,N′-dimethylethylenediamine). They were strategically designed by inserting a methyl or hydroxymethyl function at C6, to study the previously hypothesized involvement of that ring position in the anticancer properties and the peculiar metal coordination ability in aqueous solution already shown by this family of ligands. Solid state and solution studies revealed differences neither in the molecular conformation or crystal packing nor in the acid-base behavior compared with the precursor Malten. The introduced substituent groups seem to affect instead both the degradation time (ca.4-5 h forL1andL2vs.10 h for Malten) and the binding properties towards Cu(ii), Zn(ii) and Co(ii), log?Kvalues being the highest forL1within the series of the diamino-bis-maltol ligands. The introduction of -CH₂OH at C6 is sufficient to impair the biological activity of the compound and is coherent with the hypothesized mechanism of action.

Full text of DOI:10.1039/d0nj05327a

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Bis-maltol-polyamine family: structural
modifications at strategic positions. Synthesis,
coordination and antineoplastic activity of two
new ligands†
Cite this: New J. Chem., 2021,
45, 2659
a
a
a
b
Luca Giorgi,
Gianluca Ambrosi,
Daniele Paderni,
Luca Conti,
c
a
d
a
Stefano Amatori, Francesca Romagnoli, Patrizia Rossi,
Eleonora Macedi, * Claudia Giorgi,
Mauro Formica,
a
b
d
c
Paola Paoli,
Mirco Fanelli and
a
Vieri Fusi
*
0
Two new maltol-based ligands are presented, L1 (N,N -bis((3-hydroxy-6-methyl-4-pyron-2-yl)methyl)-
0
0
N,N -dimethylethylenediamine) and L2 (N,N -bis((3-hydroxy-6-hydroxymethyl-4-pyron-2-yl)methyl)-
0
N,N -dimethylethylenediamine). They were strategically designed by inserting a methyl or hydroxymethyl
function at C6, to study the previously hypothesized involvement of that ring position in the anticancer
properties and the peculiar metal coordination ability in aqueous solution already shown by this family of
ligands. Solid state and solution studies revealed differences neither in the molecular conformation or crystal
packing nor in the acid–base behavior compared with the precursor Malten. The introduced substituent
groups seem to affect instead both the degradation time (ca. 4–5 h for L1 and L2 vs. 10 h for Malten) and
the binding properties towards Cu(II), Zn(II) and Co(II), log K values being the highest for L1 within the series of
Received 30th October 2020,
Accepted 30th December 2020
DOI: 10.1039/d0nj05327a
2
the diamino-bis-maltol ligands. The introduction of –CH OH at C6 is sufficient to impair the biological activity
rsc.li/njc
of the compound and is coherent with the hypothesized mechanism of action.
are more specific and effective and less toxic (targeted thera-
pies, immunotherapies and cancer vaccines), to overcome drug
1
. Introduction
Worldwide, cancer is still one of the major leading causes of resistance and to improve conventional treatments such as
death. Much progress has been made against it over the last chemotherapy, radiation therapy and surgery.
century, nevertheless numerous challenges still prevent the
Finding new antineoplastic drugs with reduced toxicity and
possibility of providing the best outcome for patients. A WHO higher selectivity is therefore a major therapeutic goal. Natural
report claims that annual cancer cases are expected to rise from products are useful both as biological tools as well as thera-
1
14 million in 2012 to 26 million within the next 2 decades.
peutic agents and continue to be inspiring model compounds
Thus, despite the recent advances, much work remains. in medicinal chemistry.
Effort must be made to identify good molecular targets (playing In our previous studies a class of molecules has been devel-
key roles in cancer cell growth and survival) and to design and oped, constituted of a variable polyamine framework decorated
develop drugs able to bind to them, to develop treatments that with the naturally occurring substance maltol (3-hydroxy-2-
methyl-4-pyrone, Fig. 1) as a binding unit. These molecules
2
–6
combine the known acid–base properties, biocompatibility,
a
7
Dipartimento di Scienze Pure e Applicate, Universit a` degli Studi di Urbino
‘Carlo Bo’’, via della Stazione 4, I-61029 Urbino, Italy.
and antioxidant and coordination properties of maltol with the
antitumor features of linear and macrocyclic polyamines.
Such a combination could prove very fruitful indeed, since
on one hand, maltol possesses ROS scavenger and antioxidant
‘
E-mail: eleonora.macedi@uniurb.it, vieri.fusi@uniurb.it
b
c
Department of Chemistry ‘‘Ugo Schiff’’, University of Florence, via della
Lastruccia 3-13, 50019 Sesto Fiorentino, Italy
8
Department of Biomolecular Sciences, Molecular Pathology Laboratory ‘‘PaoLa’’
University of Urbino ‘‘Carlo Bo’’, via Arco d’ Augusto 2, 61032 Fano, Italy
Department of Industrial Engineering, University of Florence, via S. Marta 3,
properties that provide the molecule with antineoplastic,
9
,10
11
neuroprotective
and anti-apoptotic activities; in addition,
d
12,13
both maltol-derived organometallic complexes
and maltol-
5
0139 Florence, Italy
1
4
containing ligands have potential antitumor activity.
†
Electronic supplementary information (ESI) available: Crystallographic data are
On the other hand, rapidly proliferating cells (including cancer
cells) require a higher amount of polyamines than normal cells,
provided in PDF format. CCDC 2040416. For ESI and crystallographic data in CIF
or other electronic format see DOI: 10.1039/d0nj05327a
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Fig. 2 Coordination scheme of maltol-based ligands towards metal
cations. Green sphere: transition metal ion; blue sphere: alkaline,
alkaline-earth or rare earth metal ion.
Fig. 1 Compounds Maltol, Malten, Maltonis, L
A
, L
B
, L1 and L2.
area featuring a harder nature [binding site provided by hydroxyl
(
bridging donor groups) and carbonyl groups of maltol moieties]
and able to lodge metal cations generally recognized as difficult
thus polyamine-depleting agents might be used in cancer to bind in water, such as those of the alkaline, alkaline-earth and
15
treatment. Studies showed that synthetic polyamine analogues, rare earth series (blue sphere in Fig. 2). Mostly, heterotrinuclear
structurally similar to the biogenic polyamines without being able dimers form, where two transition metal complexes stabilize one
1
6,17
25
to functionally substitute them,
polyamine biosynthesis and to cause polyamine depletion.
are able to interfere with the hard metal cation (Fig. 2), but mononuclear and heterodinuclear
18,19
26
25b,27
monomers as discrete entities or metal coordination polymers
2
6
25b,28
Due to these features and to the relatively non-toxic response in as well as homo- and heterotetranuclear dimers
normal tissues, polyamine analogues are presently being tested in obtained.
were also
human cancer patients as potential new chemotherapeutic agents,
Following principles of drug discovery, according to which
both individually and collectively as well as in combination with the research of a new pharmacologically active molecule begins
20–22
other anticancer drugs or traditional chemotherapeutics.
from a lead compound that shows biological activity but may be
Two of the synthesized maltol-based polyamine ligands, show- improved, we have been modifying the structure of Malten
ing two [(3-hydroxy-4-pyron-2-yl)methyl]amine units separated by providing it with strategic features that can eventually help us to
0
0
either
a
linear (N,N -bis((3-hydroxy-4-pyron-2-yl)methyl)-N,N - both gain a better comprehension of the mechanism of action as
dimethylethylenediamine, Malten, Fig. 1) or cyclic (4,10-bis[(3- well as modify the biological activity of the compound.
hydroxy-4-pyron-2-yl)methyl]-1,7-dimethyl-1,4,7,10-tetraazacyclo-
To this purpose, we have recently expanded the maltol-based
dodecane, Maltonis, Fig. 1) aliphatic amine spacer, exhibited ligand family by synthesizing two Malten derivatives, N,N -
0
2
3
24
anti-neoplastic activity in vitro and in vivo.
bis((3-hydroxy-4-pyron-2-yl)methyl)-1,4-piperazine (L in Fig. 1)
A
0
0
Studies on Malten and Maltonis showed an antiproliferative and N,N ,N -tris((3-hydroxy-4-pyron-2-yl)methyl)-N-methylethy-
0
effect due to complex structural alterations of genomic DNA lenediamine (L
B
in Fig. 1) where either the linear N,N -
possibly induced by DNA intermolecular crosslinking. The dimethylethylenediamine scaffold has been replaced with the
genomic alterations lead to a dose-dependent reduction in cell cyclic piperazine (L ) or an additional maltol group has been
survival and activation of both cell cycle arrest and programmed inserted in place of a methyl group (L
A
2
9
B
). These modifications
cell death (apoptosis) as a function of the modulation in the deal with structural parameters such as stiffness, binding
expression of genes having key roles in DNA-damage response, ability and steric hindrance of the molecule. Unfortunately,
2
3,24
cell cycle progression and apoptosis.
favor the covalent binding between DNA and proteins, suggesting to the lead compound; anyway, some hints could be gained
an interference with the chromatin structure as a possible concerning their biological activity. L , notwithstanding its more
Finally, the simultaneous rigid conformation, acts by a similar mechanism to Malten, being
presence of two amino-spaced maltol units seems to be crucial however less effective. L behaves instead differently from Malten,
for the biological activity of the molecules.
being able to induce mainly single strand breaks (nicks) of DNA
Both ligands are able to such compounds do not show improved performances compared
A
2
3,24
molecular mechanism of action.
B
Malten and Maltonis also showed peculiar binding properties and, at longer time of exposure and to a lesser extent, also double
towards metal ions in aqueous solution (Fig. 2). They are able to strand breaks. Therefore, even if the structural modifications
coordinate a transition metal cation (green sphere in Fig. 2) in an brought on Malten negatively impact the biological activity, they
29
‘
‘inner’’ location (binding site provided by the polyamine scaffold result in an altered mechanism of action.
and hydroxyl groups of maltol moieties), preorganizing the
molecule, otherwise arranged in a stretched conformation,
Notwithstanding the structural modifications introduced, L
and L possess binding properties towards transition metal
in a way that maltol units converge to form a second binding ions similar to Malten, being able to form stable mononuclear
A
2
3d
B
2
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complexes. L
B
was seen to give also metal complexes with correlation experiments were performed to assign the signals.
different stoichiometries (2 : 1 and 3 : 2 metal to ligand ratio). Chemical shifts (d scale) are reported in parts per million (ppm
1
Moreover, both ligands, in particular L , are able to interact values) relative to the characteristic peak of the solvent. H NMR
A
2
9
with rare earth cations.
measurements to evaluate the stability of both L1 and L2 were
ꢀ
2ꢀ
In this work, we aim to widen the maltol-based ligand family performed at pH 7.4 in H PO /HPO4 buffer.
2
4
and study the effect of a different structural change introduced
in the molecule of Malten, in particular on the maltol ring, both
2.2 X-ray crystallography
to gain further information about the molecular mechanism of Intensity data for compound [H
2
L1]ꢁ(ClO
4
)
2
ꢁ2(H
2
O) (6) were
action as well as to investigate the already known coordination collected on an Oxford Diffraction Excalibur diffractometer
2
5
a
properties of this family towards transition metal ions. In using Mo K radiation (l = 0.71069 Å). Data collection was
3
0
particular, we followed our previous speculation on the maltol performed at 100 K. The program CrysAlis v 1.171 was used to
moiety being a Michael-type acceptor for nucleophilic sites of determine the cell parameters, to collect the intensity data and
2
3d
DNA, due to a partial positive charge at the C6 atom of maltol,
to reduce them. Intensities were corrected for Lorentz and
which may explain the ability of the lead compounds to favor the polarization effects. Absorption correction was performed with
covalent binding between DNA and proteins. To explore whether the program ABSPACK implemented in CrysAlis. The structure
3
1
such a position plays a key role in the mechanism of action, two of 6 was solved by using the SIR-2004 package and sub-
2
new maltol-based ligands decorated at C6 with two groups sequently refined on the F values by the full-matrix least-
3
2
having different dimensions were synthesized.
squares program SHELXL-2018/3. All the non-hydrogen atoms
0
Ligands L1 (N,N -bis((3-hydroxy-6-methyl-4-pyron-2-yl)methyl)- were anisotropically refined while all the hydrogen ones were
N,N -dimethylethylenediamine) and L2 (N,N -bis((3-hydroxy-6- found in the Fourier Synthesis and refined in accordance with
hydroxymethyl-4-pyron-2-yl)methyl)-N,N -dimethylethylenediamine) the atom to which they are bound. The structure was solved as a
possess, respectively, a methyl and a hydroxymethyl function at C6, twin by using the twinning law suggested by Platon. Geome-
0
0
0
3
3
3
4
that might prevent the involvement of maltol in the biological trical calculations were performed by PARST97 and molecular
3
5
mechanism, inactivating the two compounds. Furthermore, the plots were produced by the Mercury 2.4 programs. Crystal-
introduction of the hydroxyl group opens the way for the insertion lographic data and refinement parameters are reported in Table
of a fluorophore in this class of molecules, which could turn them S1 (ESI†). Deposition Number CCDC 2040416 contains the
from therapeutic to theranostic agents.
supplementary crystallographic data for this paper.†
The two ligands were synthesized and characterized both in
solution and in the solid state. The acid–base and stability proper-
2.3 EMF measurements
ties of the free ligands were investigated by potentiometric, UV-VIS Equilibrium constants for protonation and complexation reac-
and NMR measurements. The crystal structure of diprotonated L1 tions of the two ligands were determined by pH-metric measure-
ꢀ3
is reported and discussed. The coordination properties of both ments in degassed 0.15 mol dm NaCl at 298 ꢂ 0.1 K, using the
3
6
ligands in aqueous solution towards selected first row transition fully automatic equipment that has already been described. An
metal ions were also studied by potentiometric measurements. Ag/AgCl electrode in saturated KCl solution was employed as the
Finally, herein we reported the preliminary in vitro biological reference electrode, while the glass electrode was calibrated as a
studies of the free ligands. All results obtained on L1 and L2 were hydrogen concentration probe through titration of known
compared with those of the precursor Malten to highlight the amounts of HCl with CO -free NaOH solutions. EMF data were
2
3
7
influence of the structural modifications at C6 on the ligand acquired with the PASAT computer program. The equivalent
properties. point, the standard potential E and the ionic product of water
pK
o
(
w
= 13.73 ꢂ 0.01 at 298.1 K in 0.1 M NaCl) were determined by
38
using Gran’s method. At least three measurements (consisting
of 100 data points for each) were performed for each system in
the pH range of 2–11. In all experiments, the ligand concen-
2
. Experimental
2.1 General methods
ꢀ3
ꢀ3
tration ([L]) was 1 ꢃ 10 mol dm , while in complexation
UV/Vis absorption spectra were recorded at 298 K on a Varian experiments a metal ion concentration of 0.8 [L] was employed.
3
9
Cary-100 spectrophotometer equipped with a temperature control The computer program HYPERQUAD was used to process the
unit. H and C NMR spectra were acquired on a Bruker AVANCE potentiometric data. Distribution diagrams were obtained by
00 spectrometer, operating at 400.13 and 100.61 MHz for H and using the Hyss program.
1
13
1
40
4
13
C, respectively, and equipped with a variable-temperature con-
2
.4 Synthesis
troller. The temperature of the NMR probe was calibrated using
1,2-ethanediol as the calibration sample. For the spectra All chemicals were purchased in the highest quality commer-
recorded in D O, the peak positions are reported with respect cially available. The solvents were of RP grade, unless otherwise
2
1
to HOD (d = 4.75 ppm) for H NMR spectra, while dioxane was indicated.
13
used as the reference standard in C NMR spectra (d = 67.4 ppm).
For the spectra recorded in CDCl and DMSO the peak positions (30.5 g; 0.215 mol) and SOCl
are referenced to residual solvent signals. H– H and H– C sphere was stirred for 1 h. The precipitated yellow solid was
2-Chloromethyl-5-hydroxy-4H-pyran-4-one (2). A mixture of 1
3
2
(70 ml) under a nitrogen atmo-
1
1
1
13
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collected by filtration and washed with petroleum ether at were grown in RPMI 1640 (Lonza) supplemented with 10% fetal
0–70 1C, to give 2 as a white powder (30 g, 87% yield). The bovine serum (FBS), 1% penicillin–streptomycin and 1% gluta-
crude product was pure enough to be used without further mine in a humidified atmosphere at 37 1C, as previously
4
1
41
purification. H NMR (CDCl , 25 1C): d = 4.36 (s, 2H), 6.57 described. L1 and L2 were dissolved at 1 mM in distilled
3
1
3
(
s, 1H), 7.88 (s, 1H) ppm. C NMR (CDCl , 25 1C): d = 41.0, water, as working solution, immediately before use. Treatments
3
1
11.9, 138.2, 145.8, 162.9, 173.9 ppm.
were carried out at the concentrations reported in the figures
0
5-Hydroxy-2-methyl-4H-pyran-4-one (3). A solution of 2 (30 g, (cellular treatments performed at time 0 and after 24 h) and
0.187 mol) in distilled water (100 ml) was heated up to 50 1C and cellular viability was evaluated after 48 h of treatment by Trypan
stirred till the color of the solution turned pink. Zn powder (24 g, blue dye exclusion assay (using CellDropt Automated Cell
4
2
0
.375 mol) was then added, followed by the dropwise addition of Counter – DeNovix) as previously described. The data are
HCl(c), keeping the mixture at 70–80 1C. After 3 h the yellow reported as means (ꢂSD) resulting from three independent
mixture was filtered to remove the excess of Zn and then extracted experiments.
with CH Cl (5 ꢃ 200 ml). The combined organic layers were dried
2
2
2 4
(Na SO ), filtered and concentrated to give the crude product of 3
that was purified by recrystallization in 2-propanol, obtaining a 3. Results and discussion
1
light yellow powder (20.6 g, 87.5% yield). H NMR (CDCl
3
, 25 1C):
3.1 Synthetic procedures
13
d = 2.32 (s, 3H), 6.29 (s, 1H), 7.79 (s, 1H) ppm. C NMR (CDCl
3
,
0
The two compounds N,N -bis((3-hydroxy-6-methyl-4-pyron-2-
2
5 1C): d = 20.1, 111.3, 137.6, 145.3, 166.6, 174.3 ppm.
N,N -Bis((3-hydroxy-6-methyl-4-pyron-2-yl)methyl)-N,N -dimethyl-
0
0
0
0
0
yl)methyl)-N,N -dimethylethylenediamine (L1) and N,N -bis((3-
hydroxy-6-hydroxymethyl-4-pyron-2-yl)methyl)-N,N -dimethy-
lethylenediamine (L2) were obtained starting from the commercially
available 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one (Kojic acid,
in Scheme 1). The current synthetic strategy offers the advan-
tage of facile and fast preparation of the desired methylated and
hydroxymethylated compounds, avoiding the protection and
activation steps of maltol followed by SN
employed in the synthesis of Malten.
As far as L1 is concerned, the nucleophilic substitution of
the hydroxyl group of kojic acid (1) with a chloride by using
thionyl chloride gave compound 2, which was then reduced by
using Zn/HCl to give the methylated compound 3 in good yield.
The Mannich reaction of compound 3 with aqueous formaldehyde
4) and N,N -dimethylethylenediamine (5) provided ligand L1 in one
0
ethylenediamine dihydroperchlorate (L1ꢁ2HClO ). N,N -Dimethyl-
4
ethylenediamine (0.305 g, 3.5 mmol) was added under a nitrogen
atmosphere to a solution of 37% aqueous formaldehyde (0.580 ml;
1
7.8 mmol) in ethanol (2 ml). The mixture was stirred at room
temperature for 30 min, then was added dropwise to an ethanolic
solution of 3 (1.0 g, 7.1 mmol, in 15 ml EtOH). The mixture was
vigorously stirred for 5 h, then a white solid precipitated that was
filtered, washed with ethanol and dried. The crude product
was purified by dissolution in ethanol and precipitation upon
dropwise addition of a 10% perchloric acid ethanolic solution
2
conditions that were
(
0.931 g, 47% yield). (1.04 g, 47% yield, Lꢁ2HClO
4 2
ꢁ4H O).
1
H NMR (D
s, 4H), 4.41 (s, 4H), 6.39 (s, 2H) ppm. C NMR (D
d = 19.2, 40.9, 49.7, 52.3, 111.9, 140.6, 145.4, 169.0, 175.9 ppm.
2
O, 25 1C): d = 2.32 (s, 6H), 2.90 (s, 6H), 3.60
1
3
(
2
O, 25 1C):
0
(
+
step (Scheme 1a).
2 2 18
MS m/z (ESI): 365.4 (M + H ). Anal. calcd for C18H34Cl N O :
First, formaldehyde (4) is reacted with the amine scaffold 5
in ethanol to give an intermediate diiminium cation, which is
then added dropwise to an ethanolic solution of 3. Crude L1
precipitated as a white solid that was further purified by
addition of a 10% perchloric acid ethanolic solution to give
the desired compound as a yellowish dihydroperchlorate salt.
Ligand L2 can be prepared even more easily by a single one-pot
Mannich reaction starting directly from kojic acid (1), aqueous
C, 33.92; H, 5.38; N, 4.40. Found: C, 34.1; H, 5.4; N, 4.6.
0
0
N,N -Bis((3-hydroxy-6-hydroxymethyl-4-pyron-2-yl)methyl)-N,N -
0
dimethylethylenediamine (L2). N,N -Dimethylethylenediamine
(0.16 g, 1.75 mmol) was added under a nitrogen atmosphere to
a solution of 37% aqueous formaldehyde (0.32 ml; 4.30 mmol) in
ethanol (2 ml). The mixture was stirred at room temperature for
30 min, then was added dropwise to an ethanolic solution of 1
(0.5 g, 3.85 mmol, in 10 ml EtOH). The mixture was vigorously
0
formaldehyde (4) and N,N -dimethylethylenediamine (5), via for-
stirred for 5 h, then a white solid precipitated that was filtered,
1
mation of the intermediate diiminium cation (Scheme 1b). The
product is achieved as a white solid, whose purity is good enough
to allow the use of the compound as prepared. The commercial
compounds 1, 4 and 5 were used without any further purification.
washed with ethanol and dried (0.58 g, 84% yield). H NMR
(
4
DMSO, 25 1C): d = 2.21 (s, 6H), 2.55 (s, 4H), 3.57 (s, 4H), 4.28 (s,
H), 6.30 (s, 2H) ppm. C NMR (DMSO, 25 1C) d = 42.49, 53.36,
1
3
5
4.42, 60.02, 109.37, 143.92, 147.64, 167.95, 174.01 ppm. MS m/z
+
(ESI): 397.4 (M + H ). Anal. calcd for C18
24 2 8
H N O : C, 54.54; H,
3
.2 Description of the structure
Caution: Perchlorate salts of organic compounds are poten- In the asymmetric unit of [H
L1]ꢁ(ClO
tially explosive; these compounds must be prepared and handled [H L1] cation, one perchlorate anion and one molecule of
6.10; N, 7.07. Found: C, 54.4; H, 6.3; N, 7.0.
2
4
)
2
ꢁ2(H
2
O) (6) half of the
2
+
2
with care!
water are present. The two halves of the diprotonated ligand are
related by a center of symmetry (ꢀx + 1, ꢀy, ꢀz + 1).
2.5 Biological studies
As can be seen in Fig. 3, the ligand takes an open wide
The immortalized promonocytic leukemia (U937) cell line was conformation with the two aromatic rings (that lie on parallel
obtained from American Type Culture Collection (ATCC). Cells planes, due to the presence of the center of symmetry) disposed
2
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Scheme 1 Synthetic pathway for the preparation of ligands (a) L1 and (b) L2.
on opposite sides with respect to the mean plane defined by the two compounds, i.e. [H
2
L1]ꢁ(ClO
4
)
2
ꢁ2(H
2
O) (6) and [H
2
Malten]ꢁ
0
non-hydrogen atoms of the N,N -dimethylethylenediamine chain. (ClO ) ꢁ2(H O), are almost isomorphous and isostructural (see
4
2
2
The dihedral angles defining this chain have a sc/sc/ap/sc/sc below).
4
3
2+
conformation (sc: synclinal, ap: antiperiplanar) (Fig. S1, ESI†)
In contrast, the conformation of the [H L1] cation seen in
2
and the corresponding side arm orientation is o_Up-Down (Fig. S2, the studied compound is quite different from that observed for
2
3d
2+
e, ESI†). Finally the angle between the mean plane defined by the parent ligand [H
2
Malten] in the [H
2
Malten]ꢁ[PtCl
the atoms N1, C7, C7 , and N1 ( = ꢀx + 1, ꢀy, ꢀz + 1; Fig. 3) and platinum complex, where the chain connecting the two maltol
the mean plane containing the aromatic ring is 56.9(2)1. units takes a sc/ap/ap/ap/sc conformation, the overall disposition
The conformation taken by the ligand was compared with of the cation still being o_Up-Down (Fig. 5).
that of the parent compound Malten. As evidenced in Fig. 4, the In the crystal packing of 6 some strong H-bond and p–p
conformation of the diprotonated L1 ligand in 6 is well comparable interactions are present. Due to the presence of the latter, two
4
2
]ꢁ2H O
0
0
0
44
4
5
23d
2+
with that observed in [H Malten]ꢁ(ClO ) ꢁ2(H O). In addition, the symmetry related (ꢀx + 2, ꢀy + 1, ꢀz + 1) [H Malten] cations
2
4 2
2
2
are linked together and parallel chains form (the distance
between the mean planes containing the interacting rings is
3
3
.311(4) Å, while the distance between the two centroids is
.578(5) Å) (Fig. S3, ESI†).
2
+
2+
Fig. 4 Superimposition of the [H
2
L1] (ball and stick) and [H
2
Malten]
23d
(
as found in the [H
2
Malten]ꢁ(ClO
)
4 2
ꢁ2(H
2
O)
compound, stick, magenta)
2+
2
Fig. 3 Ball and stick view of the ligand cation [H L1] in 6.
cations (RMS: 0.121 Å).
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2ꢀ
species, and they can be present in solution as anionic Hꢀ2
L
species.
The analysis of the protonation constants starting from the
2
ꢀ
anionic H 2L species reveals that L1 and L2 behave very
ꢀ
similarly to each other, although L1 shows a slightly higher
log k value for each protonation step than L2. Both ligands
exhibit a quite linear decrease in basicity up to the addition of
the third proton, with logK values ranging from 9.13 to 6.98 and
8
.70 to 6.14 for L1 and L2, respectively. The last protonation step
is associated with a drop of 3 logarithmic units (logK = 3.90 and
.09, respectively); this can be justified by the electrostatic
4
3
2
+
Fig. 5 Conformation taken by [H
2
L1]
in 6 (ball and stick) and by
(stick).
repulsion occurring between the two closely spaced ammonium
2+
44
[H
2
Malten] in [H
2
Malten]ꢁ[PtCl
4
]ꢁ2H
2
O
2+
groups in the H2L species, suggesting the involvement of the
diamine moiety in this last protonation step.
The values and trend of the basicity constants are comparable
with those of the previously reported bis-maltol-diamine ligands
Table 1 Selected intermolecular hydrogen bonds in compound 6
2
3d,29
belonging to this family,
parent ligand Malten.
and in particular with those of the
D–Hꢁ ꢁ ꢁA
Dꢁ ꢁ ꢁA (Å)
Hꢁ ꢁ ꢁA (Å)
X–Hꢁ ꢁ ꢁA (1)
2
3d
O1w–H1wbꢁ ꢁ ꢁO11
3.100(5)
2.770(5)
3.049(4)
2.689(5)
3.239(6)
3.362(5)
3.385(6)
2.36(7)
2.08(6)
2.51(5)
1.73(6)
2.43(6)
2.48(5)
2.47(7)
153(7)
163(6)
126(6)
174(5)
140(4)
160(5)
167(5)
More in detail, L1 and L2 behave very similar to Malten
towards proton addition, even if values for L1 and L2 are a little
higher and a little lower, respectively, than Malten for each
protonation step. The modification introduced in the molecular
structure of Malten refers to the substituent at the carbon atom
C6 of the maltol ring, thus a positive and a negative inductive
effect of the methyl and hydroxymethyl substituents at C6 for L1
and L2, respectively, with respect to the H atom in Malten, may
O1–H1oꢁ ꢁ ꢁO11
O1w–H1wbꢁ ꢁ ꢁO2
a
N1–H1nꢁ ꢁ ꢁO2
a
C6–H6aꢁ ꢁ ꢁO1w
b
C15–H15cꢁ ꢁ ꢁO14
c
C15–H15bꢁ ꢁ ꢁO14
a
b
c
x ꢀ 1, +y, +z. ꢀx + 1, ꢀy, ꢀz + 1. ꢀx + 2, ꢀy, ꢀz + 1.
These chains are linked together by means of H-bond inter- be invoked to explain this little difference in their basicity. In
actions involving the hydrogen atom bonded to N1 and the other words, the different electronic density on the maltol ring
2
+
oxygen atom O2 of a symmetry related [H L1] cation (x ꢀ 1, caused by the presence of different groups on the C6 atom
2
+
y, +z) (see Fig. 3 for the atom labelling). Due to such interac- slightly affects the basicity of the compounds.
2
+
tions, planes perpendicular to the c axis form.
Starting from the H2L species and looking at the molecular
Finally, the water molecules and the perchlorate anions are site in each protonation step, the comparison of the basicity
located between such planes and interact with them by means constant values suggests the involvement of a nitrogen atom of
of H-bonds (see Fig. S4 (ESI†) and Table 1).
the ethylenediamine fragment in the first step (log K = 10.16 for
0
46
N,N -dimethylethylenediamine ), followed by the protonation
of the two maltolate functions (log K = 8.44 for free deproto-
3.3 Acid–base behavior
47
nated maltol ) and of the second nitrogen atom of the diamine
The acid–base behavior of L1 and L2 was investigated by both scaffold, at last.
potentiometric and UV-Vis measurements.
Fig. 6 shows the distribution diagrams of the protonated
The basicity constants of L1 and L2 were potentiometrically species of L1 and L2 together with the trend of the absorbance
determined in 0.15 M NaCl aqueous solution at 298.1 K and are at 316 and 323 nm for L1 and L2, respectively, in aqueous
summarized in Table 2. Both neutral ligands behave as diprotic solution as a function of pH (vide infra). It is to highlight that at
acids and bases under the used experimental conditions. They physiological pH = 7.4, although the neutral L species is the
2
+
are able indeed to add up to two protons, reaching the H
2
L
main one for both L1 and L2, other species coexist in solution
for both systems.
UV-Vis absorption electronic measurements in aqueous
Table 2 Protonation constants (log K) of L1 and L2 potentiometrically solution support the species suggested by potentiometric measure-
ꢀ
3
determined in 0.15 mol dm NaCl aqueous solution at 298.1 K
ments (i.e. the sites involved in the protonation), therefore allowing
to know not only the species present in solution at the different
pH values but also the acidic proton distribution exploiting the
presence of the maltol chromophore (Fig. 7).
log K
Reaction
L = L1
L = L2
2
ꢀ
+
ꢀ
a
H
H
ꢀ2
ꢀ1
L
L
+ H = Hꢀ1
L
9.13(8)
8.37(9)
6.98(7)
3.90(8)
8.70(7)
7.72(7)
6.14(9)
ꢀ
+
+
Spectrophotometrically, L1 and L2 behave very similarly to
+ H = L
2
+
+
one another, showing at pH 2, where the fully protonated H2L
L + H = HL
+
+
2+
HL + H = H
2
L
3.09(9) species is present in solution, a band with lmax = 270 and
ꢀ
1
ꢀ1
3
a
275 nm (e = 16 700 and 17 400 cm mol dm ), respectively,
Values in parentheses are the standard deviations on the last significant
figure.
that keeps lowering upon increasing the pH, while a new band
2
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that at lower energy indicates the occurrence of the deprotona-
tion of such functions.
In the case of L2, the deprotonation starts at a slightly lower
pH value than for L1 (Fig. 6), in agreement with the acidity of L2
being slightly higher than that of L1. Overall it seems that the
small structural variations introduced in the Malten molecule
to give ligands L1 and L2 do not significantly alter either the
acid–base behavior or the protonated species present in
solution at the different pH values. However, the different
groups present in the three systems on the carbon atom C6
of maltol, i.e. H in Malten, –CH in L1 and –CH OH in L2, little
3
2
affect the absorptivity in the species.
Analogously to all other members of the maltol-based ligand
family, the observed absorption behavior is in line with the
potentiometric studies and suggests the involvement of amine
functions in the first and last protonation steps, where the
2
ꢀ
+
H
ꢀ2
L
and HL species undergo the protonation; in
the remaining steps the maltolate groups are engaged in the
ꢀ
protonation, which involves the H 1L and L species to afford
ꢀ
+
the L and H L ones, respectively.
1
Potentiometric and UV-Vis data overall suggest for L1 and L2
the protonation scheme reported in Fig. 8. In the case of L1,
three species are present at a physiological pH of 7.4, with the
+
zwitterionic form L greatly prevailing on the cationic form HL
ꢀ
and on the anionic form Hꢀ1
L
(70 vs. 20 vs. 10%); in the case
of L2, two species are present at pH 7.4, with the zwitterionic
Fig. 6 Distribution diagram of the species (ꢀ) and trend of the absor-
bance (’) for (a) L1 (l = 316 nm) and (b) L2 (l = 323 nm) in aqueous
solution as a function of pH. UV-Vis measurements: [L1] = [L2] = 5 ꢃ
ꢀ
form L almost doubling the anionic form H 1L (60 vs. 35%).
ꢀ
In both cases, the zwitterionic form L can be considered as the
main species present in the biological medium at such pH,
similarly to that found for Malten.
ꢀ5
3
ꢀ3
mol dm ;
10
0
potentiometric measurements: [L1]
=
[L2]
=
1
ꢃ
ꢀ
ꢀ3
ꢀ3
1
mol dm , I = 0.15 mol dm NaCl, T = 298.1 K.
3.4 Coordination of metal cations
grows, starting from pH 5, with lmax = 316 and 323 nm, The binding properties of L1 and L2 towards Cu(II), Zn(II) and
respectively, and reaches its maximum in both cases at pH 10 Co(II) transition metal ions were investigated by potentiometric
ꢀ1
ꢀ1
3
2ꢀ
(
e = 11 200 and 8600 cm mol dm ), where the H L species measurements in 0.15 M NaCl aqueous solution at 298.1 K. The
-
2
prevails in solution (Fig. 6). The band at higher energy is related stability constants for the complexation reactions of L1 and L2
to the presence of maltol groups in their neutral form, while with the three cations are reported in Table 3, while Fig. 9
Fig. 7 Absorption spectra in the UV-Vis range of (a) L1 and (b) L2 in aqueous solution as a function of pH. [L1] = [L2] = 5 ꢃ 10ꢀ5 mol dmꢀ3
.
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Fig. 8 Location of acidic hydrogen atoms in the protonated species of L1 and L2.
2
5a,25b,25d,28,29
shows the distribution diagrams of the species for the L–M(II) and at previous studies,
a N
2
O
2
environment may
systems (L = L1, L2; M(II) = Cu(II), Zn(II), Co(II)) as a function of be suggested, coming from two oxygen atoms of the deproto-
pH. nated maltol functions of L1 and L2 along with two nitrogen
Both ligands are able to form stable mononuclear [M(H 2L)] atoms of the polyamine (Fig. 10).
ꢀ
complexes with all studied metal ions, whose formation constant
values agree with the Irving–Williams series (Table 3). Complexes form the [Cu(Hꢀ1L)] cation, with addition constant values
In both L1 and L2 the [Cu(Hꢀ2L)] species can add a proton to
+
having different stoichiometries were not found under the present ascribable to the protonation of a donor atom involved in the
experimental conditions.
Cu(II) coordination; once again, the value for the proton addi-
In all cases, the neutral [M(Hꢀ2L)] species is the one prevailing tion is slightly higher for L1 than for L2.
in solution in a wide range of pH (5–10); in some cases the pH
interval is even wider, starting at lower values (around 4, in the lated [M(H 2L)OH] species with all the tested metal ions; also
Lastly, all [M(H 2L)] species are able to form monohydroxy-
ꢀ
ꢀ
ꢀ
case of Cu(II) with both ligands) and/or ending at higher values in this case, the addition values for L1 are higher than for L2.
(around 11 in the case of Cu(II) and Co(II) with both ligands)
The comparison with Malten shows that both proton and
(
Fig. 9).
hydroxyl addition values are slightly higher for L1 than for
ꢀ
Within the series of the diamino-bis-maltol ligands (Malten, Malten except in the case of the addition of OH to the Co(II)
A
, L1, L2), L1 always provides the highest constant values for complex (log KL1 = 2.58, log KMalten = 2.92 ).
2
5a
L
the addition of Cu(II), Zn(II) and Co(II) (Table 3 and ref. 29).
These data suggest that the insertion of the electron-donor
methyl group at C6 positively affects the binding properties of
the ligand.
3
.5 Stability studies and biological data
1
H NMR experiments were performed to gain insight into the
In contrast, the insertion of the hydroxymethyl group in L2 stability of L1 and L2 in aqueous solution. Time-course analysis
produces an opposite effect compared to the methyl group, all of buffered solutions of the two ligands (phosphoric buffer, pH
stability constants being lower than those of L1 for all M(II) ions 7.4) allowed the determination of the degradation time of L1 and
1
investigated. Moreover, stability values for complex L2 fluctuate L2 thanks to the integration of peak areas in H NMR spectra. In
around those for Malten complexes.
Based on equilibrium constant values, an analogous coordi- 5 hours 30 min for L1 and L2, respectively; in both cases, the
nation environment around the M(II) ion of both ligands could degradation process is far faster than for Malten (10 h), L (10 h)
be hypothesized. Looking at the crystal structures of Malten and L (48 h).
particular, the time required for 50% degradation is 4 hours and
A
B
ꢀ3
Table 3 Logarithms of the equilibrium constants determined in 0.15 mol dm NaCl at 298.1 K for the complexation reactions of L1 and L2 with Cu(II),
Zn(II) and Co(II) cations
log K
Cu(II)
Zn(II)
Co(II)
Reaction
L = L1
L = L2
L = L1
L = L2
L = L1
L = L2
2
+
2ꢀ
a
M
[
[
+ Hꢀ2
L
= [M(Hꢀ2L)] +
19.02(8)
4.40(9)
3.31(7)
17.07(3)
3.55(3)
2.71(5)
12.31(7)
—
3.60(8)
10.44(7)
—
3.3(1)
11.75(8)
—
2.58(7)
10.36(5)
—
2.57(7)
+
M(Hꢀ2L)] + H = [M(Hꢀ1L)]
M(Hꢀ2L)] + OH = [M(Hꢀ2L)OH]
ꢀ
ꢀ
a
Values in parentheses are the standard deviations on the last significant figure.
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Fig. 9 Distribution diagram of the species for L–M(II) systems (M(II) = Cu(II), Zn(II) and Co(II)) in 1 : 1 molar ratio as a function of pH in aqueous solution.
a) L = L1; (b) L = L2. [L1] = [L2] = [Cu(II)]/[Zn(II)]/[Co(II)] = 1 ꢃ 10ꢀ3 mol dm , I = 0.15 mol dm NaCl, T = 298.1 K.
ꢀ3
ꢀ3
(
Thus, even if the introduced structural variations do not 100 mM) were conducted with L1 and L2 always in parallel to
significantly affect the acid–base properties, the substitution at the reference ligand Malten (Fig. 11).
C6 clearly makes the ligands more susceptible to degradation.
Malten, used as control was able to strongly decrease the cell
The potential of L1 and L2 to alter the biological properties survival, reaching values close to zero at the highest dose tested
2
3c,23d,29
of human cells was monitored exploiting the promonocytic (100 mM), coherently as already reported.
A different
leukemia (U937) cell line, a cellular model already used to test behavior can be observed treating the cells with L1 and L2. In
the biological properties of other maltol-based polyamine
2
3c,d,29
ligands including Malten.
In order to evaluate the
potential biological activity, treatments (at doses from 5 to
Fig. 11 Dose-dependent biological activity of L1 and L2. Cell viability of
U937 promonocytic leukemia was measured by Trypan blue dye exclusion
assay (number of Trypan blue negative cells) after 48 h of treatments.
Values are reported as means ꢂ SDs of three independent experiments.
Fig. 10 Scheme proposed for the coordination of M(II) ions (M(II) = green
sphere = Cu(II)Cu(II), Zn(II) and Co(II)) by L1 and L2 (R = CH
for L2).
3 2
, for L1; CH OH
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fact, both L1 and L2 showed a lower biological effect with Notes and references
respect to that observed with Malten, at all doses tested.
However, L1 demonstrated a more efficient biological activity
compared to L2 at higher doses (50–100 mM) (Fig. 11).
The higher effect shown by L1 with respect to L2 is in
agreement with the induction of cell death monitored at
1
2
3
M. J. Thun, J. O. DeLancey, M. M. Center, A. Jemal and
E. M. Ward, Carcinogenesis, 2010, 31, 100–110.
Joint FAO/WHO Expert Committee on Food Additives, W. H.
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R. P. H. Thompson and J. J. Powell, Aliment. Pharmacol.
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100 mM (C70% for L1 and C15% for L2). The cytotoxic effects
of Malten, instead, are induced at a dose of 50 mM (C20%) and,
more efficiently, at 100 mM (C90%).
4
5
K. H. Thompson, B. D. Liboiron, Y. Sun, K. D. D. Bellman,
I. A. Setyawati, B. O. Patrick, V. Karunaratne, G. Rawji,
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4
Conclusions
In this paper we continued our ongoing studies on maltol-
based ligands by further expanding the family through the
synthesis of two new compounds, which bear a methyl- (L1) or
hydroxymethyl substituent (L2) at the C6 position of the maltol
ring. The study was aimed at better investigating the role of
such a position in the biological mechanism of action, which is
known to involve complex structural alterations of genomic
DNA possibly induced by DNA intermolecular crosslinking.
The solid state analysis revealed that, compared to the lead
compound Malten, no differences are retrieved following the
substitution with the methyl group at C6 in terms of the
molecular conformation or crystal packing; in the same way,
solution studies disclosed an acid–base behavior similar to
Malten for both L1 and L2 ligands. However, both shorter
degradation time and better binding properties towards
Cu(II), Zn(II) and Co(II), especially in the case of L1, were found,
suggesting a possible electronic influence of the two introduced
substituent groups. From a structural point of view, the intro-
duction of a hydroxymethyl group at C6 seems sufficient to
largely impair the biological activity of the lead compound
2
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(Malten), returning a higher cell survival for L2 compared to
both L1 and Malten, perhaps following a reactivity lowering
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