6
62
L. IRALDE-LORENTE ET AL.
Synthesis compound 1
Chemical stability of 1
1
H NMR spectra were recorded after dilution of 10 mg of com-
pound 1 in a mixture of DMSO/D O 70:30. The chemical shift vari-
2
Compound 9 (120 mg, 0.318 mmol, and 1 eq) was dissolved in
a mixture of concentrated HCl (5 ml) and glacial acetic acid (5 ml).
Sn (226.5 mg, 1.908 mmol, and 6 eq) was added to the solution,
ation of the aromatic protons was used to monitor the behaviour
of 1 in solution. NMR spectra were recorded after 2 h and 24 h.
The chemical identity of compound 1 was confirmed by mass
spectrometry after 24 h in solution (MS (ESI) m/z: 363.8 [M þ H],
ꢀ
and the mixture was stirred at 80 C for 24 h before being cooled
to r.t. and filtered through celite. The solution was extracted with
AcOEt (30 ml ꢄ 3). The combined organic phases were washed
with H O and brine, and dried over anhydrous Na SO . The solv-
3
85.8 [M þ Na]).
2
2
4
ent was removed under vacuum to afford 92 mg of a white solid.
Cell viability assay
Yield: 79%
1
H NMR (400 MHz, DMSO, 298 K) (ppm): 8.22 (m, 2H); 7.78 As in a previous exercise, in vitro experiments on 14-3-3 PPI inhibi-
(
m, 1H); 7.51 (m, 2H); 7.38 (m, 3H); 4.87 (s, 2H); 3.17 (s, 3H); 2.23 (s, tors were carried out using human erythroleukemia cell line K-
13
17
3
1
1
3
H). C NMR (100 MHz, DMSO, 298 K) (ppm): 167.13, 166.48, 562 . Cancer cells were cultured in RPMI medium with 10% FCS.
61.55, 153.09, 147.40, 135.07, 133.13, 132.36, 131.39, 129.67, In order to determine antiproliferative effect of compounds, K-562
4
27.25, 124.64, 124.36, 107.04, 51.09, 36.03, 11.67. MS (ESI) m/z: cells were seeded at density of 5 ꢄ 10 cells/ml, and treated with
63.8 [M þ H], 385.8 [M þ Na]
increasing concentrations of selected compounds. Control cells
were treated with the vehicle of the experimental point contain-
ing the highest percentage of DMSO. Cell cultures were main-
ꢀ
Synthesis compound 2
tained at 37 C in 5% v/v CO for 72 h. Cell number and vitality
2
were evaluated on cell suspension using the automatic cell coun-
ter NucleoCounterVR (Chemometec, Lillerꢅd, Denmark). Each
Oxalyl chloride (0.33 ml, 3.738 mmol, and 14 eq) was slowly experiment was performed at least three times and results were
added to a solution of compound 1 (100 mg, 0.267 mmol, and expressed as mean and standard deviation (±SD).
ꢀ
1
1
eq) in THF (4 ml) at 0 C. The mixture was then stirred for
5 min, and anhydrous DMF (0.1 ml) was added dropwise. The
solution was stirred for 1 h at r.t. and then cooled again to 0 C.
MeOH was added to the reaction and left under stirring for
ꢀ
NMR spectroscopy
NMR spectra were performed at 14.1 T with a Bruker Avance 600
Spectrometer (Bruker) operating at controlled temperature (±
1
5 min. The reaction was quenched by the addition of NaOH 2 N,
and then extracted with AcOEt (15 ml ꢄ 3). The organic layer was
0
.2 K) and using a 5 mm SEI probe. Chemical shifts were refer-
washed with NaHCO , brine, dried over Na SO and evaporated
3
2
4
enced to external 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium
salt (TMSP-d4). NOESY spectra were obtained by using standard
pulse sequences. NOESY spectra were acquired with a mixing time
of 320 ms.
under reduced pressure to obtain 49 mg of a brown oil. The mix-
ture was purified by column chromatography (DCM/MeOH 95:5)
to give a yellow solid.
Yield: 47%
1
H NMR (400 MHz, DMSO, 298K) (ppm): 8.23 (m, 1H); 7.80
(
m, 1H); 7.51 (m, 2H); 7.37 (m, 3H); 4.87 (s, 2H); 3.89 (s, 3H); 3.16 (s,
H); 2.22 (s, 3H). C NMR (100 MHz, DMSO 298 K) (ppm): 166.72, Molecular modelling
66.30, 161.44, 151.07, 146.88, 134.47, 132.92, 132.37, 130.41,
29.39, 129.26, 127.16, 125.47, 124.50, 123.18, 108.11, 52.34, 50.31,
13
3
1
1
3
The crystallographic structure of 14-3-3r in complex with a phos-
phopeptide coded by PDB ID: 1YWT was used as rigid receptor in
molecular docking simulations, upon removal of the coordinates
5.86, 12.08. MS (ESI) m/z: 377.9 [M þ H], 399.8 [M þ Na]
1
8
of the phosphopeptide and crystallographic water molecules .
Docking was carried out by the GOLD docking program version
Synthesis compound 3
5
UK)
.0.1 (The Cambridge Crystallographic Data Centre, Cambridge,
1
9,20
10,11
using settings described previously
.
Borane dimethyl sulphide 1 M (0.165 ml, 0.165 mmol, and 0.6
ꢀ
eq) was added dropwise to a solution at 0 C of compound 1
(
Cells, cell culture, and transfections
100 mg, 0.275 mmol, and 1 eq) in dry DMSO. The mixture was
stirred overnight at r.t. and then quenched with K CO . The solu- HeLa cells stably expressing EGFP-Abl (HeLa EGFP-Abl) were
2
3
tion was extracted with DCM (15 ml ꢄ 3). The combined organic obtained by transfection of HeLa cells with the pCEFL EGFP ABL
2
1
layer wasw washed with HCl 1 N, brine, dried over Na
concentrated.
2
SO
4
and wild-type expression vector , using lipofectamine LTX (Life
Technologies, Carlsbad, CA), according to the manufacturer’s
instructions, followed by selection with 2 mg ml
H NMR (400 MHz, DMSO, 298 K) (ppm): 7.74 (s, 1H); 7.59 (m, Aldrich, St. Louis, MO) for 3 weeks. HeLa EGFP-Abl were main-
ꢂ 1
Yield: quantitative
G418 (Sigma-
1
2
2
H); 7.52 (m, 2H); 7.38 (m, 3H); 5.42 (s, OH); 4.77 (s, 2H); 4.63(s, tained in DMEM supplemented with 10% FBS, 2 mM L-glutamine,
1
3
ꢂ1
ꢂ1
H); 3.16 (s, 3H); 2.22 (s, 3H). C NMR (100 MHz, DMSO, 298 K) 10,000 Uml penicillin, and 10 mg ml streptomycin and were
ꢂ1
(
ppm): 167.42, 161.73, 153.18, 143.38, 141.43, 135.14, 131.83, constantly kept under selective pressure with 2 mgml
G418.
1
3
30.67, 129.65, 127.16, 124.53, 123.71, 121.35, 107.53, 62.98, 50.72, Selective medium was replaced with regular growth medium on
6.09, 11.67. MS (ESI) m/z: 371.9 [M þ Na]
the day before experiments.