Hydrophilic ligands derived from glucose: Synthesis, characterization and in vitro cytotoxic activity on cancer cells of Pt(II) complexes

Article abstract of DOI:10.1016/j.ica.2009.11.031

Aiming to contribute to the design of new antitumoral drugs, we synthesized new hydrophilic Pt(II) complexes of general formula [PtCl₂(N,N′)] containing nitrogen bidentate amine-imine and di-imine ligands derived from glucose. Some chemical properties were discussed. The X-ray molecular structure of [PtCl₂(α-d-glucopyranoside-methyl-6-deoxy-6(2-(methylimino)methyl)pyridine) (D) was reported. [PtCl₂(β-d-glucopyranosylimine-N-(2-pyridinylmethyl))] (A), which is well-soluble both in organic solvents and in water, was tested for cytotoxicity.

Full text of DOI:10.1016/j.ica.2009.11.031

Inorganica Chimica Acta 363 (2010) 741–747
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Hydrophilic ligands derived from glucose: Synthesis, characterization
and in vitro cytotoxic activity on cancer cells of Pt(II) complexes
b
b
Maria Elena Cucciolito a, Raffaella Del Litto a, , Francesco Paolo Fanizzi , Danilo Migoni ,
*
a
Giuseppina Roviello a, , Francesco Ruffo
*
a Dipartimento di Chimica, Università degli Studi di Napoli ‘‘Federico II”, Complesso Universitario di Monte S. Angelo, via Cinthia, Napoli 80126, Italy
b Dipartimento di Scienze e Tecnologie Biologiche e Ambientali (DiSTeBA), Università del Salento, Centro Ecotekne, prov. le Lecce/Monteroni, 73100 Lecce, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2009
Received in revised form 6 November 2009
Accepted 19 November 2009
Available online 26 November 2009
Aiming to contribute to the design of new antitumoral drugs, we synthesized new hydrophilic Pt(II) com-
plexes of general formula [PtCl2(N,N0)] containing nitrogen bidentate amine–imine and di-imine ligands
derived from glucose. Some chemical properties were discussed. The X-ray molecular structure of
[PtCl2(
Cl2(b-
and in water, was tested for cytotoxicity.
a
-
D
-glucopyranoside-methyl-6-deoxy-6(2-(methylimino)methyl)pyridine) (D) was reported. [Pt
D-glucopyranosylimine-N-(2-pyridinylmethyl))] (A), which is well-soluble both in organic solvents
Keywords:
Platinum
Ó 2009 Elsevier B.V. All rights reserved.
Carbohydrate
Crystal structure
Cytotoxic activity
1. Introduction
can significantly affect the solubility properties or improve the
membrane permeability.
A topical field of interest is the design of new molecules display-
ing antitumor activity. Within this ambit, platinum chemistry has
been for a long time investigated, for it offers the opportunity to
prepare very effective antitumor drugs [1]. The present research
aims to combine two features considered relevant in the design
of modern antitumor platinum compounds: the established clini-
cal efficacy of complexes with bidentate nitrogen ligands [1], and
the presence of suitable auxiliaries of natural origin, i.e. sugars,
able to confer useful physical properties such as proper solubility
in water, combined with increased membrane permeability.
In particular, interest for nitrogen chelates is demonstrated by
literature reports, which describe several Pt(II) compounds with
N,N0-diamines, N,N0-amine–imine or N,N0-di-imines ligands devel-
oped for the therapy of tumors [2].
At the same time, carbohydrates chemistry deserves growing
attention within the pharmaceutical field. In fact, sugars are often
convenient sources of chiral auxiliaries, and, hence, useful building
blocks for drugs. In addition, carbohydrates display several func-
tional groups, which can be easily functionalised. These features al-
low the synthesis of molecules with tunable properties [3]. For
example, suitable protection or deprotection of the hydroxyl groups
Currently, innovative drugs containing sugar residues, such as
bleomicine and adriamicine [2c], are used in antitumor therapy.
Advantageously, these molecules are more water-soluble than con-
ventional medicines. Furthermore, they are well tolerated by the
organism, probably because their carbohydrate fragments resem-
bles the molecules which are naturally involved in several biolog-
ical functions and which are active constituents of glycolipids,
glycoproteins and nucleotides.
On the grounds of these considerations we have developed the
synthesis of chelating nitrogen ligands (N,N0-amine–imine and
N,N0-di-imines) derived from glucose, and of the corresponding
complexes of platinum(II) [PtCl2(N,N0)] for an evaluation of their
in vitro cytotoxic activity. This strategy is expected to yield com-
pounds more featuring than the traditional drugs based on plati-
num, in terms of higher activity, reduced toxicity and easier
administration.
2. Experimental
2.1. General methods
Mono- and bi-dimensional NMR spectra were recorded in CDCl3
(CHCl3, d = 7.26; 13CDCl3, d = 77.0), CD3OD (CHD2OD, d = 3.30;
13CD3OD, d = 49.05), (CD3)2SO [(CHD2)2SO, d = 2.35; (13CHD2)2SO,
d = 39.50], by using 200, 300, 400, 500 MHz spectrometers (Varian
Model Gemini, Bruker).
* Corresponding authors. Tel.: +39 081 674459.
E-mail addresses: raffaelladellitto@inwind.it (R.D. Litto), giuseppina.roviello@u-
nina.it (G. Roviello).
0020-1693/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2009.11.031

742
M.E. Cucciolito et al. / Inorganica Chimica Acta 363 (2010) 741–747
The following abbreviations were used for describing NMR mul-
NCH2), 2.75 (dd, 1H, H2, 3J (H2–H3) = 8.3), 1.48 (s, 3H, CH3), 1.38
(s, 3H, CH3).
tiplicities: s, singlet; d, doublet; t, triplet; dd, double doublet; dt,
double triplet; m, multiplet; q, quartet. Chemical shift were re-
ported in d and coupling constants in hertz.
2.3.3. Synthesis of benzyl-2-([2-pyridinyl-methyl]amino)-2-deoxy–
D-
Specific optical rotatory powers [a] were measured with a Per-
glucoside 2-Am
kin–Elmer Polarimeter (model 141) at 298 K and 589 nm in meth-
anol, dichloromethane and dimethylsulphoxide (c = 1.0 g per
100 mL).
b3 (1.10 g, 2.74 mmol) was dissolved in aqueous acetic acid
solution (6 mL, 80% v/v) and the mixture refluxed for 2 h. Then tol-
uene was added and co-evaporated (3 ꢀ 2 mL). The product was
washed with hexane (3 ꢀ 2 mL), diethyl ether (3ꢀ2 mL) and dried
under vacuum (0.991 g, yield 86%).
The obtained product b4 (0.991 g, 2.36 mmol) was dissolved in
hot water (5 mL) and neutralized with KOH (0.230 g, 4.12 mmol).
The mixture was stirred for 30 min. The product was then ex-
tracted with dichloromethane (4 ꢀ 10 mL). The organic phases
were collected and dried over sodium sulfate. The solvent was re-
moved under vacuum and the ligand 2-Am obtained as a brown oil
(0.522 g, yield 61%).
The synthesis of b-
no-2-deoxy-4,6-O-(1-methylethylidene)-
[5], phenylmethyl 2-amino-2-deoxy- -glucopyranoside (c1) [6],
methyl 6-azido-6-deoxy- -glucopyranoside (d1) [7], cis-plati-
D-glucosamine (a1) [4], phenylmethyl 2-ami-
a-D
-glucopyranoside (b1)
a-
D
a-D
num-dichlorobis(methyl sulfoxide) [8] are described in literature.
1,4-Dioxane was distilled from Na/benzophenone.
2.2. Synthesis of [PtCl2(1-Im)] (A)
To a suspension of a1 (0.215 g, 1.20 mmol) in dioxane (4 mL),
py-2-aldehyde (0.115 mL, 1.20 mmol) was added. The addition of
[PtCl2(DMSO)2] (0.506 g, 1.20 mmol) at 298 K afforded the product
A as an orange precipitate. After 30 min of stirring, the mixture was
centrifuged, the solid washed with dioxane (2 ꢀ 3 mL) and diethyl
ether (3 ꢀ 3 mL) and dried under vacuum. Then it was further puri-
fied by column chromatography on silica gel with ethylacetate/
methanol (7/3 v/v) as eluent and the product was obtained in
62% of yield (0.400 g).
1H NMR, CDCl3, 300 MHz: d 8.50 (d, 1H, py), 7.64 (t, 1H, py),
7.40–7.15 (m, 7H, py, Ph), 4.94 (d, 1H, H1, 3J (H1–H2) = 3.3), 4.70
(t, 1H, H4, 3J (H4–H5) = 3J (H4–H3) = 10.7), 4.71(d, 1H, CHHPh,
2J = 12), 4.44 (d, 1H, CHHPh), 3.98 (qAB, 2H, NCH2, 2J = 16.0), 3.85–
3.55 (m, 4H, H3, H5, H6ax, H6eq), 2.72 (dd, 1H, H2, 3J (H2–
H3) = 10.7). 13C NMR, CDCl3, 75 MHz: d 159.5, 148.9, 137.4,
136.8, 128.3, 128.0, 127.7, 122.4, 122.1, 96.5, 73.0, 71.6, 71.0,
69.3, 62.0, 52.3. Optical activity [a] (CH3OH, 0.01 g/mL): +92.3.
1H NMR, CD3OD, 200 MHz: d 9.53 (d, 1H, py, 3J (Pt–H) = 36), 9.41
(s, 1H, N@CH, 3J (Pt–H) = 92), 8.35 (t, 1H, py), 8.16 (dd, 1H, py), 7.86
(dd, 1H, py), 5.75 (d, 1H, H1, 3J (H1–H2) = 8), 4.00–3.20 (m, 5H, H2,
H3, H4, H5, H6ax, H6eq).
2.3.4. Synthesis of B
To a solution of 2-Am (0.200 g, 0.552 mmol) in dioxane (4 mL),
[PtCl2(DMSO)2] (0.233 g, 0.552 mmol) was added. The mixture was
stirred at 323 K for 24 h. A light brown solid precipitated and it was
filtrated, washed with dioxane (3 ꢀ 5 mL) and diethyl ether
(3 ꢀ 5 mL) and dried under vacuum. The complex was obtained
as a yellow-orange product and exists as a couple of diastereoiso-
mers in 5/1 ratio (240 mg, yield 69%).
13C NMR, CD3OD, 100 MHz: d 172.3, 157.5, 149.9, 140.5, 129.5,
128.4, 91.8, 80.0, 77.6, 75.8, 69.1, 61.8. Optical activity: [
a] (CH3OH,
0.01 g/mL): +2.
Anal. Calc. for C12H16Cl2N2O5Pt: C, 26.98; H, 3.02; N, 5.24.
Found: C, 26.75; H, 3.15; N, 5.20%.
1H NMR, CD3OD/CDCl3 (5/1 v/v), 200 MHz, major diastereoiso-
mer: d 8.99 (d, 1H, py), 7.62 (t, 1H, py), 7.30–6.90 (m, 7H, py,
Ph), 6.29 (d, 1H, H1, 3J (H1–H2) = 3.2), 4.60 (d, 1H, NCHH, 2J = 13),
4.50 (qAB, 2H, CH2Ph, 2J = 16), 4.05 (dd, 1H, H3, 3J (H3–H2) = 8.5,
3J (H3–H4) = 10), 3.78 (d, 1H, NCHH), 3.70–3.30 (m, 5H, H2, H4,
H5, H6ax, H6eq). Selected resonances 1H NMR of the minor diaste-
reoisomer: d 8.92 (d, 1H, py), 5.88 (d, 1H, H1, 3J (H1–H2) = 2.8).
13C NMR, CD3OD/CDCl3 (5:1), 50 MHz: d 168.2, 147.9, 139.4,
137.9, 129.0, 128.6, 128.4, 124.0, 121.8, 99.6, 74.3, 72.1, 70.5,
2.3. Synthesis of [PtCl2(2-Am)] (B)
2.3.1. Synthesis of benzyl-2-(E-[2-pyridinyl-methylene]amino)-4,6-O-
isopropylidene-2-deoxy–D-glucoside b2
To a suspension of b1 (1.00 g, 3.23 mmol) in toluene, py-2-alde-
hyde (0.310 mL, 3.23 mmol) was added. The mixture was refluxed
for 2 h. Then the solvent was removed under vacuum and the prod-
uct obtained as an oil without further purification (1.30 g, yield
100%).
68.9, 65.7, 62.1, 56.7. Optical activity [a] (CH3OH, 0.01 g/mL):
+74.5.
Anal. Calc. for C19H24Cl2N2O5Pt: C, 36.44; H, 3.86; N, 4.47.
1H NMR, CDCl3, 200 MHz: d 8.58 (s, 1H, CH@N), 8.38 (d, 1H, py),
8.08 (d, 1H, py), 7.4–6.60 (m, 7H, py, Ph), 4.75 (d, 1H, H1, 3J (H1–
H2) = 3.4), 4.60 (d, 1H, CHHPh, 2J = 12), 4.47 (t, 1H, H3, 3J (H3–
H2) = 3J (H3–H4) = 8.3), 4.38 (d, 1H, CHHPh), 4.10 (dt, 1H, H5, 3J
(H5–H4) = 3J (H5–H6ax) = 8.3; 3J (H5–H6eq) = 4.1), 3.90 (dd, 1H,
H6eq, 2J (H6eq–H6ax) = 11), 3.75 (t, 1H, H6ax), 3.65 (t, 1H, H4),
3.45 (dd, 1H, H2), 1.52 (s, 3H, CH3), 1.38 (s, 3H, CH3).
Found: C, 36.54; H, 3.78; N, 4.68%.
2.4. Synthesis of [PtCl2(2-Im)] (C)
A solution of c1 (0.130 g, 0.490 mmol) and py-2-aldehyde
(0.047 mL, 0.490 mmol) in methanol (3 mL), was refluxed for 2 h.
Then [PtCl2(DMSO)2] (0.207 g, 0.490 mmol) was added and the
mixture was refluxed for 15 min and kept for 20 min at room tem-
perature. The slow addition of diethyl ether afforded the complex
as a brick red solid that was washed with diethyl ether and dried
under vacuum (0.199 g, yield 65%).
2.3.2. Synthesis of benzyl-2-([2-pyridinyl-methyl]amino)-4,6-O-
isopropylidene-2-deoxy–D-glucoside b3
To a cold solution of b2 (1.30 g, 3.23 mmol) in methanol/tolu-
ene 1/1 v/v (12 mL), NaBH4 (0.245 g, 6.54 mmol) was added at
273 K. After 24 h of stirring at 298 K, 25 mL of a saturated solution
of ammonium chloride was added and the product extracted with
CH2Cl2 (4 ꢀ 15 mL). The organic extracts were collected and dried
over sodium sulfate. The solvent was removed under vacuum to
get 1.10 g of the product (yield 85%).
1H NMR, CD3OD, 500 MHz: d 9.48 (d, 1H, py, 3J(Pt–H) = 39), 9.01
(s, 1H, N@CH, 3J (Pt–H) = 102), 8.32 (t, 1H, py), 8.05 (d, 1H, py), 7.83
(t, 1H, py), 7.25–7.00 (m, 5H, Ph), 5.51 (d, 1H, H1, 3J (H1–H2) = 3.4),
5.04 (dd, 1H, H2, 3J (H2–H3) = 11), 4.71(d, 1H, CHHPh, 2J = 12), 4.38
(d, 1H, CHHPh), 4.29 (t, 1H, H3, 3J (H3–H4) = 11), 3.80 (m, 1H,
H6eq), 3.70 (m, 2H, H5 e H6ax), 3.47 (m, 1H, H4).
1H NMR, CDCl3, 200 MHz: 8.38 (d, 1H, py), 7.20–6.40 (m, 8H, py,
Ph), 4.80 (d, 1H, H1, 3J (H1–H2) = 3.3), 4.55 (d, 1H, CHHPh, 2J = 12),
4.20 (d, 1H, CHHPh), 4.00–3.60 (m, 7H, H3, H4, H5, H6eq, H6ax,
13C NMR, CD3OD, 75 MHz: 171.8, 159.1, 150.7, 141.6, 138.6,
130.1, 129.6, 129.5, 129.4, 129.0, 98.2, 74.7, 72.3, 71.8, 70.7, 70.3,
62.6.

M.E. Cucciolito et al. / Inorganica Chimica Acta 363 (2010) 741–747
743
Optical activity [a] (CH3OH, 0.01 g/mL): +111.9.
Anal. Calc. for C19H22Cl2N2O5Pt: C, 36.55; H, 3.55; N, 4.49.
Table 1
Crystal, collection and refinement data for D.
Found: C, 36.49; H, 3.50; N, 4.58%.
Temperature (K)
Chemical formula
Wavelength (Å)
Crystal system
Space group
a (Å)
298
C13H18Cl2N2O5Ptꢂ2H2O
2.5. Synthesis of the complex [PtCl2(6-Im)] (D)
0.71069
monoclinic
P21
6.054(3)
6.849(4)
22.854(4)
96.19(2)
942.1(3)
2, 2.060
7.767
3.11–25.00
8771
3030 (Rint = 0.0621)
3030/228
R1 = 0.0373, wR2 = 0.0739
R1 = 0.0523, wR2 = 0.0796
0.710
In an ice cooled flask dimethylphenylphosphine (0.144 mL,
1.00 mmol) was dissolved in dioxane (5 mL) under nitrogen atmo-
sphere. In a second flask d1 (0.219 g, 1.00 mmol) was dissolved in
dioxane (5 mL) and added drop by drop to the solution of the phos-
phine. A vigorous production of nitrogen was observed. After 2 h of
stirring at room temperature py-2-aldehyde (0.096 mL,
1.00 mmol) was added and the mixture refluxed for 2 h. Then
[PtCl2(DMSO)2] (0.421 g, 1.00 mmol) was added. After 20 min, the
addition of 30 mL of diethyl ether afforded an orange solid that
was filtrated and washed with diethylether, dried under vacuum.
0.410 g of the product was obtained (yield 75%).
b (Å)
c (Å)
b (°)
V (Å3)
Z, Dx (g/cm3)
l
(mmꢁ1
)
h Range (°)
Reflections collected
Unique observed reflections
Data/parameters
R1, wR2 [I > 2r(I)]
R1, wR2 (all data)
1H NMR, DMSO-d6, 500 MHz: d 9.36 (d, 1H, py), 9.09 (s, 1H,
N@CH, 3J (Pt–H) = 98.0), 8.37 (t, 1H, py), 8.18 (d, 1H, py), 7.90 (t,
1H, py), 5.10 (d, 1H, OH, 3J = 4.9), 4.90 (br, 1H, OH), 4.80 (br, 1H,
OH), 4.47 (d, 1H, H1, 3J = 3.8), 3.98 (t, 1H, H3, 3J (H3–H4) = 3J
(H3–H2) = 10), 3.63 (t, 1H, H4, 3J (H4–H5) = 10), 3.55 (s, 3H,
OMe), 3.20 (m, 1H, H5), 3.05 (m, 1H, H2).
Largest differences in peak and hole (e Åꢁ3
)
ꢀ
ꢁ
1
P
P
2
wðF2o ꢁF2c
½
Þ
ꢃ
2
jjFojꢁjFc
jj ; wR2
¼
.
½
ꢃ
P
P
R1
¼
2
wðF2o Þ
jFo
j
13C NMR, DMSO, 75 MHz: 172.4, 156.5, 149.0, 140.7, 129.1,
128.3, 99.6, 73.2, 71.9, 68.1, 66.3, 60.8, 54.2.
Optical activity [a] (DMSO, 0.07 g/mL): +129.
Anal. Calc. for C13H18Cl2N2O5Pt: C, 28.48; H, 3.31; N, 5.11.
Found: C, 28.31; H, 3.27; N, 4.25%.
500 lg/mL. Both Pt(II) complexes and MTT were dissolved in PBS
(phosphate buffered saline). After 4 h of incubation, the amount
of formazan was spectrophotometrically measured at 550 nm
wavelength in 2-propanol. Optical density was used to calculate
cell growth inhibition, as% with respect to the control.
For the statistical analysis of the data the Bonferroni–Dunn test
was used and a p value <0.05 was considered significant. All the re-
sults are the mean of three, separately performed, experiments.
2.6. Structure determination
Yellow crystals suitable for X-ray diffraction were obtained by
slow evaporation from a methanol solution of the complex at
298 K. Data collection was performed at room temperature on a
3. Result and discussion
Bruker–Nonius kappa CCD diffractometer (Mo K
a radiation, CCD
Four new Pt(II) complexes having a N,N0-di-imine or a N,N0-ami-
no–imine ligand derived from glucose were synthesized and char-
acterized (Fig. 1) [13].
All the ligands present two nitrogen atoms in position useful to
chelate the metal. One of these functions lies on a sugar ring de-
rived from D-glucose, while the other one belongs to a pyridine
moiety. More precisely, 1-Im, 2-Im, 2-Am and 6-Im display the
imino or amino function respectively in position 1, 2 and 6 of the
rotation images, thick slices, u scans + scans to fill the asymmet-
x
ric unit). Cell parameters were determined from 37 reflections in
the range 3.056° 6 h 6 16.527°. Semiempirical absorbtion correc-
tion (multi-scan SADABS) [9] was applied. The structures were solved
by direct methods (SIR 97 package) [10] and refined by the full ma-
trix least-squares method (SHELXL program of SHELX97 package) [11]
on F2 against all independent measured reflections, using aniso-
tropic thermal parameters for all non-hydrogen atoms. All H
atoms, except for the hydroxyl protons, were positioned geometri-
cally. The hydroxyl H atoms were located in a difference Fourier
map due to the influence on their position of hydrogen bond with
the water crystallization molecules. The coordinates of these H
atoms were refined, with an isotropic displacement parameter. In
the final Fourier a small electron density (0.71 e Aꢁ3) was found
close to Pt1 atom (1.15 Å). Crystal data and details of the data col-
lection are reported in Table 1.
HO
O
HO
HO
HO
HO
OCH2Ph
N
O
N
N
NH
Pt
HO
OH
Cl
Cl Pt
Cl
Cl
2.7. Materials and methods for the cytotoxic activity
[PtCl2(2-Am)] (B)
[PtCl2(1-Im)] (A)
The MTT assay, already described by Mosmann [12], is a cyto-
toxicity test based on the metabolic reduction of a soluble tetrazo-
lium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide) (Sigma) by a mitochondrial enzyme of cultured cells into
an insoluble coloured formazan product. After harvesting, the cells
were counted and diluted appropriately with culture medium;
HO
O
N
N
HO
HO
Cl
OCH2Ph
N
O
Pt
HO
OMe
N
Cl
Cl
100 lL containing 3000 (HeLa) or 5000 (MCF-7) cells were seeded
in each well of a 96-well microtiter plate (Corning). After 24 h of
HO
OH
Pt
Cl
[PtCl2(2-Im)] (C)
incubation, cisplatin and its analogue were administered to each
well in appropriate concentrations (from 1 to 1000 lM). The toxic-
ity of these compounds was tested for 24, 48, and 72 h. At the end
of each incubation, MTT was added at a final concentration of
[PtCl2(6-Im)] (D)
Fig. 1. [PtCl2(N,N0)] complexes.

744
M.E. Cucciolito et al. / Inorganica Chimica Acta 363 (2010) 741–747
sugar ring. In all cases, the presence of free hydroxyl functions is
useful to enhance the solubility in water of the corresponding
complexes.
The complexes were obtained by reaction of the appropriate
N,N0 ligand with [PtCl2(DMSO)2]. All the novel compounds were
fully characterized by mono and bi-dimensional NMR spectroscopy
and optical measurements. The crystal structure of the complex D
was determined. The synthetic pathway for ligands and complexes
is described in Section 3.1. Cytotoxic activity of complex A was also
discussed in Section 3.2.
ligand to the metal center (d 8.53 and 9.36 ppm for the ligand and
the complex, respectively). The coupling constant to 195Pt of the
imine proton, in transoid position to the metal, is significantly lar-
ger than that of the CH@N-pyridine proton (J (Pt–H) = 92 Hz and
36 Hz, respectively), as already noted for pyridinecarboxyaldi-
mines containing aromatic groups [2d]. Complex A showed high
solubility in water (10 mg/mL) and in organic solvents (i.e.
15 mg/mL in methanol and in chloroform). In virtue of these fea-
tures, the in vitro cytotoxic activity of this complex was evaluated
(Section 3.2).
Ligand 2-Am was obtained according to Scheme 2. The amine-
sugar b1 was condensed with 2-pyridine-carboxaldehyde; the
resulting imine (b2) was reduced with sodium borohydride (b3),
then deprotected in positions 4, 6 (b4) and neutralized (2-Am).
The isolated ligand was reacted with the Pt(II) substrate, affording
the desired product B. The complexation reaction was performed
in 1,4 dioxane at 323 K, since the reaction was slower than the
precedent one (ii, Scheme 1). In these conditions, the complex
was obtained as a diasteroisomeric mixture in 5:1 ratio, as evi-
denced by 1H NMR spectrum, being different for the configuration
of the amino-nitrogen coordinated to the metal. Also complex B is
soluble in water (4 mg/mL) as well as in organic halogenated
solvents.
Similarly to 1-Im, isolation of 2-Im upon reaction of c1 with 2-
pyridine-carboxaldehyde (Scheme 3) was prevented by the possi-
ble formation of the acetal in 4, 6 position. For this reason complex
C was obtained by adding the platinum precursor in situ. The 1H
NMR spectrum showed the downfield shift of the coordinated
imine proton respect to the corresponding proton of the free ligand
(d 9.02 and 8.38 ppm, respectively) and the coupling to 195Pt of the
imine and pyridine protons in transoid and cisoid positions to the
3.1. Synthesis and characterization
Complex A was obtained in situ by adding [PtCl2(DMSO)2] to the
reaction mixture in 1,4-dioxane of 1-amino-b-D-glucopyranoside
(a1) with a stoichiometric amount of 2-pyridine-carboxaldehyde
(Scheme 1). This procedure was necessary because the reaction
of the aldehyde with the amino-sugar leads not only to the desid-
ered 1-Im ligand [14], but also to acetal a2 (evidenced by the pres-
ence of a singlet at 5.53 ppm in the 1H NMR spectrum of the crude
reaction mixture). Therefore the addition of Pt(II) substrate con-
tributed to shift the equilibrium toward the desidered product A.
The 1H NMR spectrum of the isolated compound showed a signif-
icant downfield shift for the imine proton upon coordination of the
HO
HO
HO
HO
O
O
(i)
(ii)
NH2
N
N
A
HO
OH
HO
OH
1-Im
a1
(i)
HO
HO
HO
HO
(ii)
O
O
(i)
C
OCH2Ph
OCH2Ph
N
O
O
(i) py-2-aldehyde;
(ii) [PtCl2(DMSO)2]
O
HO
NH2
HO
2-Im
N
N
NH2
c1
HO
a2
OH
(i) py-2-aldehyde;(ii) [PtCl2(DMSO)2]
Scheme 3. Synthetic pathway for C.
Scheme 1. Synthetic pathway for A.
O
O
O
O
O
O
O
O
OCH2Ph
OCH2Ph
O
OCH2Ph
(i)
(ii)
HO
N
HO HN
b3
HO
NH2
b1
b2
N
N
(iii)
HO
HO
HO
HO
O
O
(v)
(iv)
OCH2Ph
N
OCH2Ph
B
CH3COOH
HO HN
HO HN
(i) py-2-aldehyde;
(ii) NaBH4;
2-Am
b4
N
(iii) CH3COOH;
(iv) KOH;
(v) [PtCl2(DMSO)2]
Scheme 2. Synthetic pathway for B.

M.E. Cucciolito et al. / Inorganica Chimica Acta 363 (2010) 741–747
745
metal, respectively (3J (Pt–H) = 101.6 and 38.8 Hz). Complex C
shows good solubility in methanol, but poor and slow dissolution
in water.
Finally, the synthesis of D was accomplished according to
Scheme 4. Azide d1 was reacted with dimethyl phenylphosphine
affording the imine-phosphorane d2. Adding in situ the aldehyde
and, 2 h later the Pt(II) substrate, afforded complex D.
This compound shows good solubility in acetonitrile, low in
methanol, and poor in water and in organic halogenated solvents.
By slow evaporation from a methanol solution of the complex at
room temperature, single crystals suitable for X-ray characteriza-
tion were obtained. Molecular structure is reported in Fig. 2. Com-
plex D crystallizes in monoclinic system (space group P21). In the
unit cell, for each complex molecule, two crystallization water
molecules, bound through hydrogen bonds to the hydroxyl groups
of the sugar ring are present. The geometry at metal centres is typ-
ical square planar, defined by two cis chloride atoms and two nitro-
gen atoms of the imine-pyridine ligand, even if a slight deviation
from the regular coordination (N2–Pt1–N1 = 80.0(5)°) is observed,
probably due to steric constraint of the nitrogen chelate. The pyr-
idine ring is almost coplanar to the coordination plane (angle be-
tween the mean planes is 7.3(6)°). Pt–N e Pt–Cl bond distances
are typical for similar imine-pyridine Pt(II) complexes [2d]. The su-
gar ring adopts the expected chair conformation and its mean
plane is almost perpendicular to the coordination plane of the me-
tal (angle between the mean planes is 70.3(6)°; Pt1–N2–C7–
C8 = 82.6(1)°), arrangement previously reported for a palladium
complex containing a similar ligand [15].
3.2. Cytotoxic activity
Complex A was investigated by checking its in vitro cytotoxic ef-
fect on two cellular lines with respect to cisplatin: HeLa (derived
from human tumoral endometrium) and MCF-7 cells (from human
breast carcinoma), sensitive and resistant to cisplatin, respectively.
The new complex and cisplatin were administered at different con-
centrations (1, 10, 100, 200, 500, and 1000
lM) and their cytotox-
icity was measured at different incubation times (24, 48, and 72 h)
performing the MTT test.
N3
Me2PhP
N
cisplatin
complex A
(a)
(b)
(c)
O
O
(i)
100
80
HO
OMe
HO
OMe
HO
OH
HO
OH
60
d1
d2
40
(ii)
20
N
1
10
100
1000
N
Concentration (µM)
O
(iii)
HO
OMe
D
(i) PhMe2P
(ii) py-2-aldehyde
(iii) [PtCl2(DMSO)2]
HO
OH
6-Im
cisplatin
complex A
100
80
Scheme 4. Synthetic pathway for D.
60
40
20
1
10
100
1000
Concentration (µM)
cisplatin
complex A
100
80
60
40
20
1
10
100
1000
Concentration (µM)
Fig. 2. ORTEP-3 view of the complex D. Thermal ellipsoids are shown at 30%
probability level. Selected bond distances and angles (Å, °): Pt1–Cl1 = 2.294(3),
Fig. 3. The sensitivity of HeLa cells to Pt(II) complexes. Cells were treated with
increasing concentrations of complex A and cisplatin; viable cell number was
determined after 24 h (a), 48 h (b), and 72 h (c) of incubation by MTT assay,
respectively. The data are the means SD of three different experiments run in
eight replicates and are presented as percent of control.
Pt1–Cl2 = 2.296(3),
Pt1–N1 = 2.006(9),
Pt1–N2 = 2.002(8),
N1–C5 = 1.38(1),
C5–C6 = 1.42(2), N2–C6 = 1.27(2), N2–Pt1–N1 = 80.0(5), N2–Pt1–Cl2 = 95.7(4), Cl2–
Pt1–Cl1 = 90.6(1), N1–Pt1–Cl1 = 93.8(3), N1–C5–C6–N2 = 2.3(2), Pt1–N2–C7–
C8 = 82.6(1).

746
M.E. Cucciolito et al. / Inorganica Chimica Acta 363 (2010) 741–747
After 24 h incubation on HeLa cells complex A showed a much
lower cytotoxic effect with respect to cisplatin, reaching an IC50 va-
lue (concentration required for 50% growth inhibition) of
The different biological effect of the two molecules was also ob-
served at longer incubation times (48 and 72 h), especially in the
1–500
complex A with respect to cisplatin (IC50 values at 48 and 72 h
were 692.3 20.8 M and 684.8 34.2 M for the new synthe-
sized compound; 6.6 0.1 M and 4.7 0.1 M for cisplatin). At
the highest tested dose (1000 M) cell survival reached 18% and
lM concentration range, giving a lower cytotoxicity for
893.9 35.8
l
M whereas cisplatin gave the same cytotoxic effect
M (Fig. 3a and Table 2). The new platinum com-
M concen-
M cisplatin already resulted in a 70%
depletion of viable cells. The highest in vitro cytotoxicity was ob-
tained at the 1000 M dose: 43% of cell survival for complex A
at 59.1 1.8
l
l
l
pound exhibited a negligible cytotoxicity in the 1–500
l
l
l
tration range whereas 100
l
l
21% for complex A and only 7% and 5% for cisplatin, after 48 and
72 h, respectively (Fig. 3b and c, and Table 2).
l
and only 10% for cisplatin (Fig. 3a).
The biological assays on MCF-7 cells showed after 24 h in the 1–
500 lM concentration range a very low and comparable cytotoxic
effect for both the new synthesized Pt complex and cisplatin,
although complex A IC50 value was significantly higher (a value
Table 2
The IC50 values (concentration required for 50% growth inhibition of cell cultures) of
complex A and cisplatin on HeLa and MCF-7 cells, calculated after 24, 48, and 72 h of
incubation. The data are the means SD of three different experiments.
>1000
lM for the new complex and 833.4 33.3 lM for cisplatin,
Fig. 4a and Table 2).
At longer incubation times (48 and 72 h) the differences be-
tween the new complex and cisplatin became more evident. In
24 h
48 h
72 h
Cisplatin
Complex A
Cisplatin
59.1 1.8
893.9 35.8
833.4 33.3
>1000
6.6 0.1
4.7 0.1
684.8 34.2
9.7 0.5
HeLa
the 1–500
gible toxicity, whereas 100
and 80% depletion of viable cells, after 48 and 72 h, respectively
(IC50 values at 48 and 72 h were 800.0 24.0 and
719.6 54.0 for the new synthesized compound and
M and 9.7 0.5 M for cisplatin, Fig. 4b and c, and Ta-
lM concentration range the complex A exhibited negli-
692.3 20.8
41.9 1.3
800.0 24.0
lM cisplatin already resulted in a 70%
MCF-7
Complex A
719.6 54.0
lM
l
M
41.9 1.3
ble 2).
l
l
cisplatin
complex A
In conclusion the complex A was characterized by higher cyto-
toxicity on HeLa cells with respect to MCF-7 cells, as observed for
cisplatin although with a much lower effectiveness. Indeed the IC50
values measured on HeLa cell cultures were all significantly lower
than the corresponding IC50 values measured on MCF-7 cells both
for the new compound and cisplatin, but new complex IC50 values
were much higher with respect to cisplatin IC50 (Table 2). This re-
sult was confirmed by the time-course analysis of the cytotoxic ef-
fect of the complex A and cisplatin at the highest tested dose
100
80
60
40
20
1
10
100
1000
Concentration (µM)
(1000 lM, Fig. 5) where for HeLa cells the new complex caused
ꢄ60% depletion after 24 h and ꢄ80% after 48 and 72 h whereas cell
(a)
survival was negligible at every incubation time for cisplatin. The
cisplatin
complex A
administration of 1000 lM to MCF-7 cells caused depletion of
the cell cultures ꢄ50% after 24 h, ꢄ65% after 48 h and ꢄ75% after
72 h in the case of the complex A and ꢄ60% after 24 h, ꢄ85% after
48 h and ꢄ90% after 72 h in the case of cisplatin.
100
80
60
40
20
4. Conclusion
New square–planar Pt(II) complexes containing ligands derived
from glucose were prepared and fully characterized. Complex A
displayed higher cytotoxicity on HeLa cells with respect to MCF-
1
10
100
1000
Concentration (µM)
(b)
cisplatin
complex A
c
c
i
o
s
m
p
p
l
pp
la
lle
a
tin
n
HeeLLaa
ex A
100
80
100
80
60
40
20
c
c
i
o
s
m
p
p
l
pp
la
lle
a
tin
n
MCFF--77
ex A
60
40
20
1
10
100
1000
Concentration (µM)
24
48
Time (h)
72
(c)
Fig. 4. The sensitivity of MCF-7 cells to Pt(II) complexes. Cells were treated with
increasing concentrations of complex A and cisplatin; viable cell number was
determined after 24 h (a), 48 h (b), and 72 h (c) of incubation by MTT assay. The
data are the means SD of three different experiments run in eight replicates and
are presented as percent of control.
Fig. 5. Time course of the cytotoxic effect of complex A and cisplatin, administered
at 1000 M concentration, on HeLa and MCF-7 cells. The data are the means SD of
three different experiments run in eight replicates and are presented as percent of
control.
l

M.E. Cucciolito et al. / Inorganica Chimica Acta 363 (2010) 741–747
747
7 cells, as observed for cisplatin, although with a much lower effec-
References
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worthy of further investigation, aiming to disclose possible alter-
native benefits, such as minor toxicity side effects or easier admin-
istration with respect to other widely used platinum drugs.
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CCDC 725069 contains the supplementary crystallographic data
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Supplementary data associated with this article can be found, in
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