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cis-[Pt(NH ) (naph)Cl]NO (1): Compound 1 was prepared accord-
ing to an already reported procedure with slight modifications.
Experimental Section
3
2
3
[19]
Chemistry
A solution of cisplatin (150 mg, 0.5 mmol) in N,N-dimethylforma-
mide (DMF; 5 mL) was treated with AgNO3 (84.9 mg, 0.5 mmol),
and the reaction mixture was stirred in the dark for 16 h at 558C.
The AgCl precipitate was removed by filtration, and 1,8-naphthyri-
dine (58.5 mg, 0.9 equiv) was added to the filtrate. The reaction
mixture was stirred for 16 h at 558C. The resulting brown solution
was filtered, concentrated in vacuo to a minimum volume and di-
Materials and methods: Commercial reagent grade chemicals
(
e.g., glutathione, guanosine 5’-monophosphate (5’-GMP), 1-
methyl-cytosine) and solvents were used as received without fur-
ther purification. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT) and oxaliplatin were obtained from Sigma
[
27]
luted with MeOH. Addition of Et O induced the formation of
Chemical (St. Louis, MO, USA). Cisplatin
and [PtI(Me phen)-
2
2
[11,28]
a brown precipitate that was isolated by filtration, washed with
Et O and ice cold H O, and dried in vacuo (108 mg, 0.224 mmol,
(
MeCY)]I
were prepared according to reported procedures.
2
2
1
1
195]
H NMR and [ H- Pt] HSQC spectra were recorded on a Bruker
1
4
5%): H NMR (CD OD): d=9.46 (dd, J=5.32, 1.73 Hz, 1H, CH ),
3 2
1
5
1
13
Avance DPX 300 MHz instrument. 1D N NMR, 2D TOCSY, [ H, C]-
HSQC, and [ H, N]-HSQC NMR spectra were recorded on a Bruker
Avance III 700 MHz instrument. H and C chemical shifts were ref-
erenced to the residual solvent peak (3.30 and 49.15 ppm for
9
.25 (dd, J=4.23, 1.92 Hz, 1H, CH ), 8.70 (dd, J=8.12, 1.73 Hz, 1H,
7
1
15
CH ), 8.58 (dd, J=8.12, 1.92 Hz, 1H, CH ), 7.81 (dd, J=8.12, 4.25 Hz,
4
5
1
13
13
1
H, CH ), 7.71 ppm (dd, J=8.12, 5.33 Hz, 1H, CH ); C NMR
6 3
(
CD OD): d=160.30 (C2), 156.23 (C7), 142.27 (C4), 139.77 (C5),
3
1
13
15
CD OD, respectively for H and C). N chemical shifts were refer-
15
3
125.31 (C6), 124.52 ppm (C3).
N NMR (D O): d=ꢀ67.88,
2
1
5
enced to external NH Cl (1m in HCl 1m) placed at 0 ppm with re-
spect to liquid NH3.
[195]
4
195]
ꢀ69.96 ppm. Pt NMR (CD OD): d=ꢀ2243 ppm; Anal. calcd for
3
[
Pt NMR spectra were referenced relative to
C H ClN O Pt·1.5H O: C 19.86, H 3.13, N 14.48, found: C 19.87, H
8
12
5
3
2
[29]
external K PtCl placed at ꢀ1620 ppm with respect to Na [PtCl ]).
+
2
4
2
6
2.79, N 14.12; MS (ESI): m/z: 395.03 [M] .
1
1
D H NMR measurements for kinetic studies were performed on
1
5
15
15
cis-[Pt( NH ) (naph)Cl]NO ( N-1). Compound N-1 was prepared
a Bruker Avance III 700 MHz instrument. NMR spectra were collect-
ed at different reaction times and processed with identical settings.
Selected signals of the aromatic amine ligand were automatically
integrated with Bruker TopSpin 3.0 software and integral values
were used to calculate the relative concentration of the corre-
sponding platinum complex at each point.
3 2
3
using the same procedure reported for 1 starting from cis-[PtCl -
2
1
5
(
NH ) ]. The elemental analyses and the spectroscopic characteris-
3 2
1
5
tics of N-1 were consistent with the data obtained for 1.
Reactivity of compound 1: The aquation of compound 1 was in-
vestigated in a 2 mm D O solution at 378C (dioxane (3.75 ppm)
2
[30]
was used as internal standard).
Electrospray ionisation mass spectrometry (ESI-MS) was performed
with a dual electrospray interface and a quadrupole time-of-flight
mass spectrometer (Agilent 6530 Series Accurate-Mass Quadrupole
Time-of-Flight (Q-TOF) LC-MS).
15
The reaction of N-1 with 5’-GMP was performed at 378C in NMR
tubes containing a 6.57 mm solution of the complex (90:10 H O/
2
D O, 31.3 mm PBS buffer, pH 7.2*) and of 5’-GMP (16 equiv,
2
1
05 mm). Trimethylsilylpropionic acid (TSP) was used as internal
Elemental analyses were carried out with a Eurovector EA 3000
CHN instrument.
standard.
1
5
The reaction of compound N-1 with glutathione (GSH) was per-
formed at 378C in an NMR tube containing a 6.23 mm solution of
A Crison MicropH meter Model 2002 equipped with Crison micro-
combination electrodes (5 and 3 mm diameter) and calibrated with
Crison standard buffer solutions at pH 2.00, 4.01, and 7.02 was
used for pH measurements. pH values marked with an asterisk (*)
of the NMR samples are the measured pH values without correc-
tion for the effect of deuterium on the pH meter electrode.
the complex (90:10 H O/D O, 65.0 mm PBS buffer, pH 7.4*) and
2
2
GSH (1 equiv). TSP was used as internal standard.
Biology
1
,8-naphthyridine (naph): This compound was prepared accord-
Compounds 1, 2, cisplatin, and oxaliplatin were dissolved in
a 0.9% NaCl solution just before the experiment.
[21]
ing to the method of Paudler and Kress with modifications.
Sodium 3-nitrobenzensulfonate (40 g, 0.178 mol) was dissolved in
concd H SO (50 mL), and the resulting solution was treated with
anhyd glycerol (25 g, 0.271 mol) and stirred vigorously to obtain
homogeneity. The resulting mixture was treated with 2-aminopyri-
dine (7.5 g, 0.08 mol) and stirred in an oil bath at 1308C for 5 h,
then cooled in an ice bath and made alkaline by addition of concd
NaOH. The alkaline solution was then extracted with CHCl3 (4ꢁ
Cell cultures: Human pancreatic (BxPC3), lung (A549), colon (HCT-
15, DLD1 and LoVo) carcinoma cell lines, along with melanoma
(A375), were obtained from American Type Culture Collection
(ATCC, Rockville, MD, USA). Human cervical carcinoma cells A431
were kindly provided by Prof. F. Zunino (Division of Experimental
Oncology B, Istituto Nazionale dei Tumori, Milan, Italy). The LoVo-
OXP cells were derived, using a standard protocol, by growing
LoVo cells in increasing concentrations of oxaliplatin and following
13 months of selection of resistant clones, as previously de-
2
4
1
00 mL). The combined CHCl fractions were extracted with aq HCl
3
(
4ꢁ100 mL; pH 3.0, 42 mL of concd HCl in 0.5 L H O). The com-
2
bined aqueous fractions were adjusted to pH 5 with concd NaOH,
and the solution was extracted with CHCl (4ꢁ100 mL). The various
CHCl fractions were combined, dried over anhyd MgSO , filtered,
and evaporated to dryness in vacuo. The crude solid residue was
[31]
scribed. Cell lines were maintained in the logarithmic phase at
378C in 5% CO2 using various culture media (RPMI-1640, F-12
Ham’s, DMEM) containing 10% fetal calf serum (Euroclone, Milan,
3
3
4
ꢀ
1
ꢀ1
purified by sublimation at 808C to give 1,8-naphtyridine (2 g,
Italy), antibiotics (50 unitsml penicillin and 50 mgmL streptomy-
cin) and 2 mm l-glutamine. RPMI-1640 medium (Euroclone) was
used for BxPC3, HCT-15, A431 and DLD1 cells, F-12 Ham’s (Sigma
Chemical Co.) was used for A549, LoVo and LoVo-OXP cells, and
Dulbecco’s modified Eagle’s medium (DMEM; Euroclone) was used
for A375 cells.
1
2
7
1
5
2
0%): H NMR (CD OD): d=9.08 (dd, J=4.23, 1.95 Hz, 2H, CH ),
.67 (dd, J=8.17, 1.26 Hz, 2H, CH ), 8.48 ppm (dd, J=8.17,
.90 Hz, 2H, CH ); C NMR (CD OD): d=154.75 (C2/7), 139.41 (C4/
), 123.84 ppm (C3/6); Anal. calcd for C H N : C 73.83, H 4.65, N
3
2,7
3
,6
1
3
4
,7
3
8
6
2
1.52, found: C 73.95, H 4.72, N 21.40.
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1161 – 1168 1167