J.-R. Pallandre et al. / European Journal of Medicinal Chemistry 103 (2015) 163e174
171
A saved for docking. Docking of the filtered set of the BioinfoDB
database was done using standard “speed1 settings” of the PLANTS
docking program [43] and the Chemplp scoring function.
iodide (1.153 g, 7.69 10ꢀ3 mol) and sodium carbonate (815 mg,
8.3 mmol) in dry acetone. The reaction mixture was stirred to
reflux. Overnight and then it was diluted with water and extracted
three times with ethylacetate. The combined organic layer was
washed with brine, dried over sodium sulfate, filtered and evapo-
rated under vacuum. The product was obtained by flash chroma-
tography (cyclohexane e dichloromethane: 4e6) as a white solid
(788 mg, 85%). IR (ATR): 3051, 3026, 2920, 2838, 2741, 1691, 1589,
1572 cmꢀ1. 1H NMR (300 MHz, CDCl3) ppm 9.83 (s, 1H), 7.48 (s, 1H),
7.39 (d, J ¼ 7.7 Hz, 2H), 7.35 (s, 1H), 7.17 (d, J ¼ 7.7 Hz, 2H), 5.13 (s,
2H), 3.93 (s, 3H), 2.35 (s, 3H). 13C NMR (75 MHz, CDCl3) ppm 189.9,
154.5,149.6,138.1,133.5,132.4,129.3,129.0,128.6,125.8,109.4, 75.0,
56.2, 21.2.
Hit selection was based on a two-step knowledge-based pro-
tocol aimed at identifying compounds that share phosphorylated
peptide key interactions with key residues of Stat3. For this pur-
pose, all docking poses were converted into 1D protein-ligand
interaction fingerprints (IFP) using the in-house developed IFP
program [12]. A first reference IFP (IFP1: 4 bits/residue) was defined
between pTyr705 (Stat3 B chain) and its anchoring residues
(Lys591, Arg609) in chain A, considering polar interactions only
(hydrogen bonds and salt bridges). A second IFP (IFP2: 7 bits/res-
idue) was then defined between the phosphorylated peptide
Ala703-Pro704-pTyr705-Leu706-Lys707 of chain B and its full binding
site (Lys591, Arg595, Arg609, Ser611, Ser613, Ser614, Thr620,
Phe621, Trp623, Lys626, Ile634, Gln635, Ser636, Val637, Glu638,
Pro639, Tyr640, Tyr657, Ile659, Cys712, Val713, Thr714, Pro715,
Phe716) in chain A considering all interactions (hydrophobic, aro-
matic, hydrogen bonds and salt bridges) [12]. Tanimoto coefficients
were then computed between the interaction fingerprints of all
BioinfoDB docking poses and each of the two reference IFPs. Poses
with a Tanimoto coefficient of 1.0 (IFP1) and higher than 0.5 (IFP2)
were selected next, leaving a final set of 52 non-redundant hits.
4.2.2. N-(3-chloro-5-methoxy-4-(4-methylbenzyloxy)benzyl)-2H-
tetrazole-5-amine 5
A solution of 42 (154 mg, 0.53 mmol) and 5-aminotetrazole
(90 mg, 0.11 mmol) in a mixture of methanol and dichloro-
methane (a suspension was prepared in methanol and dichloro-
methane was added until dissolution) was stirred at rt for 2 h. Then
sodium cyanoborohydride (40 mg, 0.64 mmol) was added and the
reaction mixture was further stirred overnight. The reaction was
quenched with water. The product was extracted with ethylacetate.
The combined organic layers were washed with brine and dried
over sodium sulfate. The product was obtained by column chro-
matography (dichloromethane e methanol: 95-5) as a white solid
(76 mg, 40%). IR (ATR): 3253, 3146, 3051, 2923, 2880, 1624, 1578,
1537, 1518 cmꢀ1. 1H NMR (300 MHz, DMSO D6) ppm 14.65 (s, 1H),
7.53 (t, J ¼ 6.2 Hz, 1H), 7.34 (d, J ¼ 7.8 Hz, 2H), 7.18 (d, J ¼ 7.8 Hz, 2H),
7.05 (d, J ¼ 1.1 Hz, 1H), 6.98 (d, J ¼ 1.1 Hz, 1H), 4.90 (s, 2H), 4.34 (d,
J ¼ 6.2 Hz, 2H), 3.84 (s, 3H), 2.31 (s, 3H). 1H NMR (300 MHz, DMSO
D6) ppm 153.4, 152.1, 142.2, 137.1, 136.1, 133.9, 128.6, 128.2, 126.7,
119.7, 110.9, 73.9, 56.0, 46.1, 20.7. HRMS (ESI) calcd for
4.1.3. Molecular dynamics simulations and ensemble docking
The structure of human STAT3 monomer was constructed by
homology modeling using the full-length sequence and the human
STAT1 crystal structure 1BF5 as a template. The homology modeling
was performed with the SWISS- MODEL server [44]. The model
obtained was subject to all-atom molecular dynamics simulations in
water in the NPT ensemble at a temperature of 300 K and a pressure
of 1 atm. GROMACS 4.6.5 software suit with GROMOS 54A7force
field was used. The protein was energy minimized and subject to
20 ns simulation with restrained backbone followed by unrestrained
60 ns production simulation. The equilibration was monitored by the
RMSD of Ca atoms. The last 10 ns of the trajectory were used to
extract 200 frames at 5 ps intervals. These frames were used as the
representative structures in ensemble docking simulations.
The docking procedure was performed as follows. The structures
of selected inhibitors were optimized using Gaussian09 at the
B3LYP/6e31þþG(d) level of theory and their ESP partial charged
were computed. The ligands and the corresponding protein struc-
tures were prepared for docking using the MGLTools-1.5.6. The
docking was performed with the Autodock Vina [45]. Analysis of
the docking results was performed with custom software based on
the Python binding of the Pteros molecular modeling library [46].
C
17H18ClN5O2Na 382.1047 [(M þ Na)þ]; found 382.1053.
4.2.3. 3-chloro-5-methoxy-4-(4-nitrobenzyloxy)benzaldehyde 46
The 4-nitrobenzyl bromide (1.19 g, 5.51 mmol) was added to a
suspension of the 5-chlorovanilline (1.03 g, 5.51 mmol), and sodium
carbonate (876 mg, 8.3 mmol) in dry DMF. The reaction mixture
was stirred at rt overnight and then it was diluted with water and
extracted three times with ethylacetate. The combined organic
layer was washed with brine, dried over with sodium sulfate,
filtered and evaporated under vacuum. The product was obtained
by recrystallization (cyclohexane e dichloromethane: 4e6) as a
white fluffy solid (1.5 g, 84%). IR (ATR): 3107, 3085, 3008, 2950,
2868, 1697, 1606, 1593, 1574, 1517 cmꢀ1. 1H NMR (300 MHz, DMSO
D6)
d
ppm 9.91 (s, 1H), 8.27 (d, J ¼ 7.2 Hz, 2H), 7.78 (d, J ¼ 7.2 Hz, 2H),
4.2. STAT inhibitors synthesis
7.68 (s, 1H), 7.56 (s, 1H), 5.30 (s, 2H), 3.95 (s, 3H). 13C NMR (75 MHz,
DMSO D6) 191.0, 153.8, 148.0, 147.1, 144.3, 132.7, 128.7, 127.6, 124.0,
123.4, 111.0, 73.0, 56.4.
Starting material reagents and analytical grade solvents were
purchased from SigmaeAldrich and Acros. All reactions were
routinely checked by TLC using Merck Kieselgel 60 F254 aluminum
plates and visualized by UV light. IR spectra were performed on a
Spectrum65 perkin Elmer with UATR and principal absorptions are
given in cm-1. 1H and 13C NMR spectra were recorded in the
specified deuterated solvent at 300 MHz and 75 MHz on a Brucker
AC 300 instrument. Chemical shifts are expressed in parts per
4.2.4. N-(3-chloro-5-methoxy-4-(4-nitrobenzyloxy)benzyl)-2H-
tetrazole-5-amine 23
A solution of 46 (1.03 g, 3.20 mmol) and 5-aminotetrazole
(545 mg, 6.41 mmol) in a mixture of methanol and dichloro-
methane (a suspension was prepared in methanol and dichloro-
methane was added until dissolution) was stirred at r.t. for 2 h.
Then sodium cyanoborohydride (242 mg, 3.84 mmol) was added
and the reaction mixture was further stirred overnight. The reac-
tion was quenched with water. The product was extracted with
ethylacetate. The combined organic layers were washed with brine
and dried over with sodium sulfate. The solvent was removed in
vacuum and the product was obtained by recrystallization (cyclo-
hexane e dichloromethane:4e6) as a white solid (700 mg, 56%). IR
(ATR): 3274, 3161, 3076, 2940, 2885, 2850, 2779, 2694, 1643, 1602,
million (d) relative to the solvent signal and the coupling constants J
are given in Hertz (Hz). ESI e MS analyses were carried out at the
Service Commun d'Analyse, ICMR e UMR CNRS 6229e51,100
Reims.
4.2.1. 3-chloro-5-methoxy-4-(4-methylbenzyloxy)benzaldehyde 42
The 4-methylbenzyl chloride (540 mg, 3.84 mmol) was added to
a suspension of the 5-chlorovanilline (596 mg, 3.20 mmol), sodium