Modeling, synthesis and NMR characterization of novel chimera compounds targeting STAT3

Article abstract of DOI:10.1039/c4md00177j

Signal Transducer and Activator of Transcription 3 (STAT3) is a cytoplasmic factor that mediates intracellular signaling commonly generated at cell surface receptors and transmits it to the nucleus. In previous research studies we synthesized several mole

Full text of DOI:10.1039/c4md00177j

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Modeling, synthesis and NMR characterization of
novel chimera compounds targeting STAT3†
Cite this: Med. Chem. Commun., 2014,
5, 1651
Silvia Dell'Orto,a Daniela Masciocchi,a Stefania Villa,a Fiorella Meneghetti,a
a
a
b
bc
*
Giuseppe Celentano, Daniela Barlocco, Diego Colombo, Laura Legnani,
Lucio Toma,c Yoon Jung Jeon,d Byoung-Mog Kwon,d Akira Asaie
a
*
and Arianna Gelain
Signal Transducer and Activator of Transcription 3 (STAT3) is a cytoplasmic factor that mediates intracellular
signaling commonly generated at cell surface receptors and transmits it to the nucleus. In previous research
studies we synthesized several molecules inhibiting STAT3 and among them oxadiazole MD77 and
pyridazinone I showed an interesting activity. For the first one, a direct inhibition mechanism was
determined, while I interfered within the STAT3 pathway at a different level. In order to discover novel
compounds possibly endowed with an improved activity, we decided to merge their scaffolds on the
basis of their calculated conformational properties. Therefore we designed and synthesized the chimera
diastereomers 3-oxo-N-(4-(trifluoromethyl)phenyl)-2,3,4,4a,5,6-hexahydrobenzo[h]cinnoline-6-
carboxamide (1, 2) and 3-oxo-N-(4-(trifluoromethyl)phenyl)-2,3,5,6-tetrahydrobenzo[h]cinnoline-6-
carboxamide (3) as racemate and their enantiomeric separation was performed. Modeling studies and
theoretical prediction of [a]D values were carried out beside NMR studies which allowed us to assign 1
and 2 relative configurations.
Received 18th April 2014
Accepted 30th July 2014
DOI: 10.1039/c4md00177j
www.rsc.org/medchemcomm
In previous research studies focused on the discovery of new
inhibitors targeting aberrant STAT3 signaling, we described the
oxadiazole MD77,3 a direct STAT3 SH2 domain inhibitor (IC50
value 17.7 mM, by Alpha-Screen based assay) and the pyr-
idazinone I,4 exhibiting good inhibitory activity (46% STAT3
inhibition at 2 mM concentration, by dual luciferase assay), but
unable to bind the SH2 domain (Chart 1).
Docking studies performed on MD77 showed that, in the best
scored pose of the STAT3–MD77 complex, it maintained the same
geometry shown to be preferred in water solution, established by
molecular modeling calculations, and characterized by torsional
angles s1 ꢀ s4 more or less deviated from planarity.3 In this pose
MD77 showed a series of interactions with suitable functional
groups in the SH2 domain involving its various molecular
portions, among which the p-triuoromethylphenyl moiety
Introduction
STATs (Signal Transducers and Activators of Transcription) are
latent cytoplasmic proteins transducing signals through the
cytoplasm and acting as transcription factors in the nucleus,
regulating cell growth and survival.1 They are constituted by
several structurally and functionally conserved domains: among
them, the Src homology 2 (SH2) domain is essential for the
STAT activation cascade. STAT3, a member of the STAT family,
has been found constitutively activated in a wide variety of
human solid and blood tumors and the suppression of
deregulated STAT3 activation leads to cancer cell apoptosis with
tumor regression, slightly affecting normal cells.2
a
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Milano, Via L.
Mangiagalli 25, 20133 Milano, Italy. E-mail: arianna.gelain@unimi.it; Fax: +39-02-
503-19359; Tel: +39-02-503-19369
b
`
Dipartimento di Biotecnologie Mediche e Medicina Traslazionale, Universita degli
Studi di Milano, Via Saldini 50, 20133 Milano, Italy
c
`
Dipartimento di Chimica, Universita degli Studi di Pavia, Via Taramelli 12, 27100
Pavia, Italy. Fax: +39-0382-98-7323; Tel: +39-0382-98-7311
dLaboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience
& Biotechnology and Department of Biomolecular Science, Korea University of Science
and Technology, Eoun-Dong, Yuseong-gu, DaeJeon 305-333, South Korea
eCenter for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of
Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4md00177j
Chart 1
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Chart 2
Scheme 1 Reagents and conditions: (a) PPA, 100/110 ꢁC, 30 min; (b)
4-(trifluoromethyl)aniline, CH2Cl2, TBTU, N-methlymorphine upto
pH ¼ 7, rt, 24 h; (c) CHO–COOH, NaOH, H2O, EtOH, rt, 1 h; (d) Zn,
CH3COOH, H2O, rt, 20 min; (e) NH2NH2$H2O, EtOH, reflux, 2 h; (f)
Sodium 3-nitrobenzenesulfonate, NaOH, H2O, reflux, 30 min.
engaged in several hydrogen bonds. As far as pyridazinone I is
concerned, an important requisite for inhibitory activity versus
STAT3 seems to be the planar or almost planar arrangement of
the polycyclic system, that presents only one geometry signi-
cantly populated, in complete agreement with that described in a
previous paper and obtained with a molecular mechanics
approach.5
The main degree of conformational freedom in I is repre-
sented by the N2 side chain, able to assume two almost iso-
energetic orientations, that, however, does not seem to exert a
signicant effect on the activity.
condensed with 4-(triuoromethyl)aniline, in the presence of
the coupling agent TBTU (O-(benzotriazol-1-yl)-N,N,N0,N0-tetra-
methyluronium tetrauoborate), to obtain the amide 5. Aldol
condensation between 5 and glyoxylic acid, followed by dehy-
dration, led to the a,b-unsaturated acid 6.
Taking into account the biological results of these previously
studied molecules, and their geometrical features determined
by molecular modeling calculations, we have chemically
merged their scaffolds aided by molecular modeling calcula-
tions in order to identify new molecules endowed with
enhanced activity against STAT3. Their synthesis was planned
to verify if the MD77 p-triuoromethylphenyl moiety, which
plays a crucial role for the binding to the target, could address
the pyridazinone nucleus in proximity of the dimerization
interface. This approach started from the above stated consid-
eration concerning the almost planar conformation of the
tricyclic moiety. Therefore, the chimeras 1–3 (Chart 2) were
designed introducing on the central ring of I the p-tri-
uoromethylphenyl moiety of MD77 in a position able to
maintain its original orientation within the protein binding
pocket.
Reduction of the double bond by zinc in acetic acid gave a
mixture of the two diastereoisomers 7 and 8, which were not
separated and directly cyclized with hydrazine monohydrate to
give the diastereoisomers 1 and 2 which were separated by ash
chromatography. Dehydrogenation of the dihydropyridazinonic
ring of 1 was carried out with sodium 3-nitrobenzenesulfonate
to give 3 while 2 did not react under the same conditions. The
relative congurations of 1 and 2 were assigned by means of
NOESY experiments.
Finally, in order to perform the biological evaluation of the
single enantiomeric entities, we separated the racemates 1, 2
and 3 by means of a chiral semi-preparative HPLC.
Computational studies
The modeling studies on the diastereoisomers 1 and 2, and on
compound 3 were carried out with the Gaussian09 program
package using the B3LYP exchange–correlation functional at
the 6-311+G(d,p) level.6 The studies were performed on the
stereoisomers 1a, 2a and 3a. The discussion is referred to them
but it also applies to their enantiomers 1b, 2b and 3b, whose
conformers are mirror images of those of 1a–3a. The degrees of
conformational freedom were considered, namely inversion of
Here we report the synthesis of 1–3, the complete exploration
of their conformational space performed through theoretical
calculations, the racemate separations, the NMR characteriza-
tion as well as the results of the biological assays.
Chemistry
Starting from the commercially available a-phenylglutaric the central hexacyclic ring and rotation along the single bond
anhydride, by treatment with polyphosphoric acid, the racemic connecting the amide carbonyl group to this ring, with energy
acid 4 was obtained (Scheme 1). The carboxylic group of 4 was minimization and optimization of all the possible starting
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geometries. Aer the optimization in vacuo, a further optimi- resembles 1a in the preference for the pseudo-axial orientation,
zation was performed at the same level as above using a though in a lesser percentage (89.4%).
polarizable continuum solvent model (PCM), choosing chloro-
Then, in an attempt to suggest the absolute conguration of
form, dimethyl sulfoxide, or methanol as the solvent, for 1a, 2a these compounds, the specic optical rotation of each
and 3a, respectively. Fig. 1 shows the minimum energy conformer of compounds 1a–3a was predicted using DFT
conformations of each compound together with the corre- calculations at the same level as above.7 The calculated [a]D for
sponding relative energy and the percentage contribution to the each conformer was weighted averaged on the basis of its
overall population at 298 K. A very different conformational percentage contribution to the overall population and yielded a
behaviour was found between the two diastereoisomers 1a and positive value for 2a as well as for 3a ([a]D ¼ +375 and +387,
2a. In fact, whereas in 1a the triuoromethylphenylamido chain respectively) whereas the value obtained for 1a was too low for a
almost exclusively (99.9%) prefers the pseudo-axial orientation, reliable prediction. The largely positive values predicted for 2a
in its diastereoisomer 2a the opposite pseudo-equatorial and 3a, compared with the largely positive rotation values of the
orientation is largely preferred (96.2%). Compound 3a closely rst eluted enantiomer of 2 and the second eluted enantiomer
of 3 (see Experimental), allows us to suggest that the former has
the (6R,4aR) conguration and the latter the (6R) conguration
whereas no prediction seems possible for the enantiomers of 1.
1H-NMR studies
Racemic compounds 1, 2 and 3 were submitted to high eld NMR
spectroscopy (see Experimental) in order to get experimental data
on their 3D structure. In particular for each compound 1H reso-
nances were assigned and coupling constants measured. They
are reported in Tables 1 and 2 together with the theoretical
chemical shis and coupling constants calculated for each
Table 1 Experimental and calculated selected 1H NMR chemical shifts
(ppm) of compounds 1, 2 and 3
H
1 Exp.a
1 Calcd
2 Exp.b
2 Calcd
3 Exp.c
3 Calcd
6
3.99
1.89
2.84
3.01
—
3.83
1.71
2.85
2.80
—
3.97
1.95
2.29
2.98
—
3.52
2.07
2.08
2.69
—
4.09
3.20
3.26
—
6.83
—
3.56
2.93
3.47
—
6.78
—
5axd
5eqd
4a
4
4axd
4eqd
2.23
2.71
2.00
2.47
2.36
2.52
2.16
2.47
—
—
a
CDCl3, 298 K. b DMSO-d6, 308 K. c CD3OD, 298 K. d The indexes ax and
eq are referred to the most stable conformer of each compound.
Table 2 Experimental and calculated selected 1H NMR coupling
constants (Hz) of compounds 1, 2 and 3
J
1 Exp.a 1 Calcd 2 Exp.b 2 Calcd 3 Exp.c 3 Calcd
6,5ax
6,5eq
5ax,5eq 13.0
5ax,4a
5eq,4a
5ax,4
5eq,4
4a,4ax
4a,4eq
4.8
2.3
5.1
2.5
11.8
11.1
4.8
—
—
13.4
6.1
12.5
4.0
12.5
12.5
4.0
—
—
16.0
6.6
10.2
3.8
11.6
10.4
4.9
—
—
13.5
6.1
5.4
4.8
16.4
—
6.1
2.9
14.5
—
13.0
5.0
—
—
16.0
6.5
—
—
1.8
1.0
—
2.2
0.5
—
—
—
Fig.
1 Three-dimensional plots of the populated conformers of
4ax,4eq 16.0
15.2
16.0
15.1
—
—
compounds 1a (1a-A,B,C), 2a (2a-A,B,C,D) and 3a (3a-A,B,C,D,E) with
CDCl3, 298 K. b DMSO-d6, 308 K. c CD3OD, 298 K.
a
their relative energy (kcal molꢀ1) and population percentages.
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assay8 in human colorectal carcinoma cells HCT-116, charac-
terized by uncontrolled expression of STAT3. The results
are expressed as percentage inhibition aer 24 h treatment
with the test compounds and Cryptotanshinone9 (a natural
phenanthrene-quinone derivative) which was used as the
control (Table 3).
At the lower concentration none of the compounds showed
any signicant activity, while at 50 mM concentration the best
derivative was (+)-1, which however was less active than cryp-
totanshinone (35% and 85%, respectively). Also, the
AlphaScreen-based assay10 did not reveal any strong interaction
of the chimeras with the STAT3 SH2 domain (% inhibition < 5),
though they are incorporating the triuoromethyl moiety that,
in the case of MD77, cooperates in the binding to this domain.
Conclusion
During our studies, which aimed at the discovery of new
potential STAT3 inhibitors, MD77 and I emerged as interesting
molecules able to interfere in a different manner within the
STAT3 pathway. These compounds were chemically merged
leading to the chimeras 1, 2 and 3, by utilizing the moieties
identied as important for the STAT3 activity.
Fig. 2 Chemical structures of compounds 1a and 2a and their cor-
responding preferred conformers. In the 3D-plot of 2a a double arrow
highlights the proximity of H-6 and H-4a that explains the NOESY
observed cross-peak.
The computational analysis on 1–3 revealed a very different
conformational behavior of these compounds. In fact, while in 1
the triuoromethylphenylamido chain almost exclusively
prefers the pseudo-axial orientation, in 2 the opposite pseudo-
equatorial orientation is largely preferred. Compound 3 closely
resembles 1 in the preference for the pseudo-axial orientation,
though in a lesser percentage. This geometrical diversity
encouraged the synthetic efforts, in particular the orientation of
the p-triuoromethylphenyl group with respect to the tricyclic
moiety in 2 that seems comparable with the orientation of the
same group with respect to the chlorophenyl oxadiazole moiety
in MD77. Thus, the chimeras were synthesized and their race-
mate resolved.
Then, high eld NMR spectroscopy and NOESY experiments
were performed to give experimental support to the calculations
and unambiguously assign the 1 and 2 relative congurations.
The vicinal J6,5ax coupling constant of 1 and 2 (Table 2) is in
complete agreement with, respectively, an axial and equatorial
orientation of the triuoromethylphenylamido chain. The
combination of the observed J6,5 and J5,4a in the two diastereo-
isomers is compatible with the assignment of the (6R,4aS)/
(6S,4aR) relative conguration to 1 and the (6R,4aR)/(6S,4aS) to
2. Moreover, the intense cross-peak between H-6 and H-4a,
observed only in the NOESY spectrum of 2, supports again its
relative conguration, that makes these two hydrogens oriented
optimized conformation of 1a–3a and averaged on the basis of
the population percentages reported in Fig. 1.
NOESY experiments were also performed to conrm the 1
and 2 relative congurations. In fact for the (6R,4aR)/(6S,4aS)
˚
conguration (Fig. 2B), a 2.6 A calculated distance between the
C-6 and C-4a protons should account for a NOE contact. On the
contrary, this should not be observable for the (6R,4aS)/(6S,4aR)
˚
conguration (Fig. 2A) that showed a 3.8 A calculated distance.
Actually, the comparison of the NOESY spectrum of 1 with the
corresponding spectrum of 2 (see Fig. S1a and S1b, ESI† section)
clearly showed an intense cross peak only between H-6 and H-4a
of 2 whose conguration was therefore ascertained to be
(6R,4aR)/(6S,4aS). Consequently 1 resulted to be unambiguously
the (6R,4aS)/(6S,4aR) isomer.
Biological evaluation
The STAT3 inhibitory activity was evaluated at 10 mM and 50 mM
concentrations through a modied procedure of dual luciferase
Table 3 Dual luciferase assay results of compounds 1–3
Compound
% inh. [10 mM]
% inh. [50 mM]
˚
on the same face of the molecule and relatively close (2.6 A from
1
(+)-1
ꢀ3
9
9
ꢀ6
ꢀ8
1
6
the theoretical calculations) and not the diastereoisomeric
(6R,4aS)/(6S,4aR) conguration, that makes them trans-oriented
35
14
3
12
16
7
24
30
85
(ꢀ)-1
2
˚
and more spaced (3.8 A).
(+)-2a
Finally, both the racemates and each single enantiomer of
the new compounds were submitted to biological evaluation
that did not bring about the identication of an advanced
chemical lead, in spite of the presence in the molecules of
moieties that have been shown suitable in other substrates.
(ꢀ)-2b
3
2
(+)-3a
ꢀ2
(ꢀ)-3b
2
Cryptotanshinone
78
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This indicates that the portions of MD77 not included in the with a saturated solution of sodium hydrogencarbonate (1 ꢂ 2
chimera compounds seem to play a specic role that probably mL) and nally with brine (1 ꢂ 2 mL). The organic phase was
should not be neglected.
dried over anhydrous Na2SO4, ltered and concentrated in
vacuo. The residue was puried by ash chromatography
(cyclohexane–ethyl acetate 7 : 3) to afford the intermediate 5
(62% yield). 1H NMR (CD3OD) 2.43–2.48 (m, 2H, CH2), 2.64
(ddd, 1H, J ¼ 17.5, J ¼ 6.0 and J ¼ 6.0 Hz, CH), 2.96 (ddd, 1H, J ¼
Experimental
General
Unless otherwise noted the materials were obtained from 17.5, J ¼ 14.0 and J ¼ 6.2 Hz, CH), 4.13 (dd, 1H, J ¼ 5.7 and J ¼
commercial suppliers (as starting reagents reported in 5.7 Hz, CH), 7.36 (dd, 1H, J ¼ 7.50 and J ¼ 1.0 Hz, ArH), 7.42
Scheme 1) and used without purication. Commercial plates on (ddd, 1H, J ¼ 7.50, J ¼ 7.50 and J ¼ 1.0 Hz, ArH), 7.57 (ddd, 1H, J
aluminium-backed Silica Gel 60 plates (0.2 mm, Merck) were ¼ 7.50, J ¼ 7.50 and J ¼ 1.5 Hz, ArH), 7.60 (d, 2H, J ¼ 8.6 Hz,
used for analytical TLC to follow the course of the reaction and ArH), 7.80 (d, 2H, J ¼ 8.6 Hz, ArH), 8.03 (dd, 1H, J ¼ 7.50 and
to check the product purity. Silica gel 60 (Merck 40–63 mM) was J ¼ 1.5 Hz, ArH).
used for ash chromatography to purify intermediates and nal
(Z)-2-(1-Oxo-4-(4-(triuoromethyl)phenylcarbamoyl)-3,4-dihy-
compounds. The purity of nal compounds was determined by dronaphthalen-2(1H)-ylidene)acetic acid 6. A solution of NaOH
HPLC analysis and was $95%. Melting points were determined (0.26 g, 6.53 mmol) in water (3.1 mL) and ethanol (1.63 mL) was
on a Buchi 510 capillary melting point apparatus and are added dropwise to an ice-cooled solution of compound 5 (0.44 g,
uncorrected. 1H NMR spectra were recorded on a Bruker 1.33 mmol) and glyoxylic acid (0.49 g, 5.27 mmol) in water (1.8
AVANCE-500 spectrometer operating at 500.13 MHz; the mL). This solution was stirred at room temperature for 1 h and
chemical shis are reported as d (ppm) using the solvent as the then ethanol was removed under reduced pressure. The
internal standard. The enantiomeric separations of compounds obtained aqueous mixture was acidied with 6 N HCl and the
1 and 3 were performed by a chiral semi-preparative HPLC precipitate was ltered, washed with 6 N HCl (1 ꢂ 1 mL) and
Chiralcel ODH (5 mm, 250 ꢂ 4.6 mm). The enantiomeric sepa- dried to give a yellow residue. The crude solid was puried by
ration of compound 2 was performed by a chiral semi-prepar- ash chromatography (dichloromethane–methanol 9 : 1 with
1
ative HPLC Chiralpak AD (5 mm, 250 ꢂ 4.6 mm). Optical 0.02 mL of acetic acid) to provide compound 6 (47% yield). H
rotation values of the enantiomers were registered on a Perkin NMR (CD3OD) 3.16 (ddd, 1H, J ¼ 16.6, J ¼ 4.8 and J ¼ 2.7 Hz,
ꢁ
Elmer instrument (Mod 343) at 589 nm and 25 C. The struc- CH), 4.13 (dd, 1H, J ¼ 16.6 and J ¼ 4.0 Hz, CH), 4.22 (dd, 1H,
tures of all compounds were consistent with their analytical and J ¼ 4.8 and J ¼ 4.0 Hz, CH), 6.91 (br s, 1H, ¼CH), 7.46 (dd, 1H,
spectroscopic data.
J ¼ 7.50 and J ¼ 1.0 Hz, ArH), 7.48 (ddd, 1H, J ¼ 7.80, J ¼ 7.50 and
4-Oxo-1,2,3,4-tetrahydronaphthalene-1-carboxylic acid 4. A J ¼ 1.1 Hz, ArH), 7.56 (d, 2H, J ¼ 8.5 Hz, ArH), 7.61 (ddd, 1H, J ¼
mixture of a-phenylglutaric anhydride (0.5 g, 2.69 mmol) and 2 7.50, J ¼ 7.50 and J ¼ 1.4 Hz, ArH), 7.70 (d, 2H, J ¼ 8.5 Hz, ArH),
g of polyphosphoric acid was heated at 100–110 ꢁC for 15 min. 1 8.07 (dd, 1H, J ¼ 7.8 and J ¼ 1.4 Hz, ArH).
g of polyphosphoric acid was added and the mixture was heated
2-(1-Oxo-4-(4-(triuoromethyl)phenylcarbamoyl)-1,2,3,4-tetra-
for 15 min at the same temperature. The solution appeared as a hydronaphthalen-2-yl)acetic acids 7, 8. Compound 6 (0.173 g,
dense brown oil. The mixture was cooled with an ice bath, 0.44 mmol) and zinc dust (0.34 g, 0.52 mmol) in acetic acid (4.95
treated with water and extracted with ethyl acetate (3 ꢂ 2 mL). mL) and water (1.24 mL) were heated at 60 ꢁC for 20 min. Then
The organic phase was rstly treated with 1 N NaOH (2 mL) and the reaction mixture was cooled to room temperature and the
then the collected alkaline aqueous solution was acidied with amount of zinc was ltered off. The mixture of diastereoisomers
6 N HCl (1 mL) and extracted with ethyl acetate (3 ꢂ 1 mL). The 7 and 8 (91% yield) was extracted with ethyl acetate (3 ꢂ 2 mL);
organic layer was dried over anhydrous Na2SO4 and evaporated the organic layer was dried over anhydrous Na2SO4 and the
under reduced pressure to afford 4 as a brown oil (90% yield). solvent was evaporated under reduced pressure. The mixture was
1H NMR (CDCl3) 2.38 (dddd, 1H, J ¼ 13.8, J ¼ 12.0, J ¼ 4.8 and J not separated and used without further purication. 1H NMR
¼ 4.8 Hz, CH), 2.52 (dddd, 1H, J ¼ 13.8, J ¼ 4.8, J ¼ 4.8 and J ¼ (CD3OD) 2.36 (ddd, 1H, J ¼ 12.7, J ¼ 12.7 and J ¼ 12.7 Hz, 1H,
4.8 Hz, CH), 2.65 (ddd, 1H, J ¼ 17.4, J ¼ 4.8 and J ¼ 4.8 Hz, CH), CH), 2.47 (ddd, 1H, J ¼ 12.7, J ¼ 4.3 and J ¼ 4.3 Hz, 1H, CH), 2.51
2.92 (ddd, 1H, J ¼ 17.4, J ¼ 12.0 and J ¼ 4.8 Hz, CH), 3.99 (dd, (dd, 1H, J ¼ 16.8 and J ¼ 6.4 Hz, CH), 2.95 (dd, 1H, J ¼ 16.8 and J
1H, J ¼ 4.8 and J ¼ 4.8 Hz, CH), 7.37 (dd, 1H, J ¼ 7.50 and J ¼ 1.0 ¼ 5.7 Hz, CH), 3.13 (dddd, 1H, J ¼ 12.7, J ¼ 5.7, J ¼ 6.4 and J ¼ 4.3
Hz, ArH), 7.39 (ddd, 1H, J ¼ 7.50, J ¼ 7.50 and J ¼ 1.0 Hz, ArH), Hz, CH), 4.27 (dd, 1H, J ¼ 12.0 and J ¼ 4.3 Hz, CH), 7.38–7.43 (m,
7.53 (ddd, 1H, J ¼ 7.50, J ¼ 7.50 and J ¼ 1.5 Hz, ArH), 8.06 (dd, 2H, ArH), 7.57 (ddd, 1H, J ¼ 7.50, J ¼ 7.50 and J ¼ 1.5 Hz, ArH),
1H, J ¼ 7.50 and J ¼ 1.5 Hz, ArH).
7.64 (d, 2H, J ¼ 8.5 Hz, ArH), 7.86 (d, 2H, J ¼ 8.5 Hz, ArH), 8.02
4-Oxo-N-(4-(triuoromethyl)phenyl)-1,2,3,4
tetrahydro- (dd, 1H, J ¼ 7.8 and J ¼ 1.5 Hz, ArH).
naphthalene-1-carboxamide 5. To a solution of compound 4 (0.2
3-Oxo-N-(4-(triuoromethyl)phenyl)-2,3,4,4a,5,6-hexahydro-
g, 1.05 mmol) in dichloromethane (6 mL) the coupling agent benzo[h]cinnoline-6-carboxamide 1, 2. To a mixture of diaste-
TBTU (0.46 g, 1.42 mmol), N-methylmorpholine (up to pH ¼ 7) reoisomers 7 and 8 (0.15 g, 0.38 mmol) in ethanol (4 mL),
and 4-(triuoromethyl)aniline (0.4 mL, 3.21 mmol) were added. hydrazine monohydrate (0.09 mL, 1.93 mmol) was added
The resulting mixture was stirred at room temperature for 24 h. dropwise and the obtained yellow solution was reuxed for 2 h.
The product was extracted with dichloromethane (3 ꢂ 2 mL). Aer evaporation of the solvent, the reaction mixture was
The organic layer was washed rst with 1 N HCl (1 ꢂ 2 mL), then extracted with ethyl acetate (5 ꢂ 1 mL). The organic layer was
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dried over anhydrous Na2SO4, ltered and the solvent was broadband reverse probe. Chemical shis are reported on the
evaporated under reduced pressure. The two diastereoisomers 1 d (ppm) scale and are relative to chloroform (7.24 ppm), DMSO
and 2 (64% yield; diastereomeric ratio 1 : 2 ¼ 8 : 2) were sepa- (2.51 ppm) or methanol (3.30 ppm) signals. Compounds (about
rated by ash chromatography (dichloromethane–methanol 5 mg) were dissolved in 0.5 mL of CDCl3 (1), DMSO-d6 (2) or
9.8 : 0.2). Compound 1 (yellow solid): mp 230.2–232.4 ꢁC. 1H CD3OD (3) under N2, and their assignments were given by a
NMR (CDCl3) 1.89 (ddd, 1H, J ¼ 13.0, J ¼ 13.0 and J ¼ 4.8 Hz, combination of 1D and 2D COSY experiments, using standard
CH), 2.23 (dd, 1H, J ¼ 16.0 and J ¼ 16.0 Hz, CH), 2.71 (dd, 1H, J Bruker pulse programs. Z-PFGs were used to obtain 1H–1H
¼ 16.0 and J ¼ 6.5 Hz, CH), 2.84 (ddd, 1H, J ¼ 13.0, J ¼ 5.0 and J COSY spectra. The pulse width was 7.50 ms (90ꢁ) for 1H. Typically
¼ 2.3 Hz, CH), 3.00 (dddd, J ¼ 16.0, J ¼ 13.0, J ¼ 6.5 and J ¼ 5.0 32 768 data points were collected for one-dimensional spectra.
Hz, 1H, CH), 3.99 (dd, 1H, J ¼ 4.8 and J ¼ 2.3 Hz, CH), 7.10 (br s, The spectral width was 11.45 ppm (5733 Hz) for 1H NMR (digital
1H, NH), 7.34 (dd, 1H, J ¼ 7.0 and J ¼ 1.9 Hz, ArH), 7.42–7.52 (m, resolution: 0.17 Hz per point). 2D experimental parameters
1
6H, ArH), 8.23 (dd, 1H, J ¼ 7.4 and J ¼ 1.9 Hz, ArH), 8.52 (br s, were as follows. For H–1H correlations: relaxation delay 2.0 s,
1H, NH). The enantiomeric separation of 1 was performed by a 1024 ꢂ 1024 data point matrices (512 experiments to 1024 zero
chiral semi-preparative HPLC: Chiralcel ODH (5 mm, 250 ꢂ 4.6 lling in F1, 1024 in F2), 2 or 16 transients in each experiment
mm), hexane–ethanol (85 : 15), 0.8 mL minꢀ1, UV 254 nm; tR(1) for COSY and NOESY respectively, spectral width 11.15 ppm
¼ 10.564 min (49.92%). (+)-1: [a]2D5 ¼ +16 (c 0.0016, CH3OH); (5580.37 Hz). The NOESY spectra were generated with a mixing
tR(2) ¼ 12.503 min (50.08%) (ꢀ)-1: [a]2D5 ¼ ꢀ16 (c 0.0016, time of 1.0 s and acquired in the TPPI mode. There were no
CH3OH). Compound 2 (white solid): mp (277.1–279.8 ꢁC). 1H signicant differences in the results obtained at different mix-
NMR (DMSO-d6) 1.95 (ddd, 1H, J ¼ 12.5, J ¼ 12.5 and J ¼ 12.5 ing times (0.5–1.5 s). All 2D spectra were processed with the
Hz, 1H, CH), 2.29 (ddd, 1H, J ¼ 12.5, J ¼ 4.0 and J ¼ 4.0 Hz, CH), Bruker soware package.
2.36 (dd, 1H, J ¼ 16.0 and J ¼ 16.0 Hz, CH), 2.52 (dd, 1H, J ¼ 16.0
and J ¼ 6.6 Hz, CH), 2.98 (dddd, J ¼ 16.0, J ¼ 12.5, J ¼ 6.6 and J ¼
Computational methods
4.0 Hz, 1H, CH), 3.97 (dd, 1H, J ¼ 12.5 and J ¼ 4.0 Hz, CH), 7.16
All calculations were performed using the Gaussian09 program
(dd, 1H, J ¼ 7.20 and J ¼ 1.0 Hz, ArH), 7.34 (ddd, 1H, J ¼ 7.80, J ¼
7.20 and J ¼ 1.0 Hz, ArH), 7.37 (ddd, 1H, J ¼ 7.20, J ¼ 7.20 and
J ¼ 1.8 Hz, ArH), 7.73 (d, 2H, J ¼ 8.5 Hz, ArH), 7.92 (d, 2H, J ¼ 8.5
Hz, ArH), 8.08 (dd, 1H, J ¼ 7.8 and J ¼ 1.8 Hz, ArH). The
enantiomeric separation of 2 was performed by a chiral semi-
preparative HPLC: Chiralpak AD (5 mm, 250 ꢂ 4.6 mm), hexane–
ethanol (85 : 15), 0.8 mL minꢀ1, UV 254 nm; tR(1) ¼ 14.970 min
(49.68%). 2a: [a]2D5 ¼ +264 (c 0.0012, DMSO); tR(2) ¼ 19.781 min
(50.32%). 2b: [a]D25 ¼ ꢀ287 (c 0.0012, DMSO).
package,6 at the B3LYP/6-311+G(d,p) level. Theoretical values of
the [a]D were obtained at the same level of theory specifying
appropriate keywords (polar and cphf) into the Gaussian input.
GIAO NMR11 calculations were carried out at the B3LYP/6-
311+G(d,p) level. The coupling constants were calculated with
Ramsey's nonrelativistic approach12 and were weight-averaged
on the basis of the population percentages.
Biological assays
3-Oxo-N-(4-(triuoromethyl)phenyl)-2,3,5,6-tetrahydro-benzo[h]-
cinnoline-6-carboxamide 3. A mixture of compound 1 (0.050 g, 0.13
Dual luciferase assay. Cell culture: the cancer cell lines were
mmol), sodium m-nitro-benzenesulfonate (0.029 g, 0.13 mmol), obtained from American Type Culture Collection. Human
and NaOH (0,021 g, 0.52 mmol) in water (1 mL) was reuxed for 30 breast cancer cell lines (MDA-MB-468, MDA-MB-231) and
min. Aer cooling, the reaction mixture was extracted with ethyl human colon cancer cell line (SW620) were maintained in RPMI
acetate (3 ꢂ 1 mL) and the organic layer was dried over anhydrous 1640 (Gibco/BRL). Human colon cancer cell line (HCT-116) was
Na2SO4, ltered and the solvent was evaporated under reduced maintained in McCoy's 5A (Gibco/BRL). All culture media were
pressure. The obtained residue was puried by ash chromatog- supplemented with 10% heat-inactivated fetal bovine serum
raphy (dichloromethane–methanol 9.7 : 0.3) to afford 3 as a yellow (Gibco/BRL). Cell cultures were maintained at 37 ꢁC under a
solid (85% yield). Sublimation was performed at 333.4–338.7 ꢁC. 1H humidied atmosphere of 5% CO2 in an incubator. Transient
NMR (CD3OD) 3.20 (ddd, 1H, J ¼ 16.4, J ¼ 5.4 and J ¼ 1.8 Hz, CH), transfection and dual-luciferase assays:8 HCT-116 cells were
3.26 (ddd, 1H, J ¼ 16.4, J ¼ 4.8 and J ¼ 1.0 Hz, CH), 4.09 (dd, J ¼ 5.4 seeded at a density of 10 ꢂ 105 cells in 100 mm2 culture plate.
and J ¼ 4.8 Hz, 1H, CH), 6.83 (br s, 1H, CH), 7.38–7.44 (m, 3H, ArH), The cells were co-transfected with pSTAT3-TA-Luc (27 mg per
7.57 (d, 2H, J ¼ 8.5 Hz, ArH), 7.71 (d, 2H, J ¼ 8.5 Hz, ArH), 8.14 (m, plate) and internal control plasmid pRL-TK (9 mg per plate)
1H, ArH). The enantiomeric separation of compound 3 was per- containing the Renilla luciferase gene. All plasmids used in this
formed by a chiral semi-preparative HPLC: Chiralcel ODH (5 mm, experiment were purchased from Promega. The transfection
250 ꢂ 4.6 mm), hexane–ethanol (80 : 20), 0.8 mL minꢀ1, UV 254 was carried out using TransFectin (Bio-Rad) according to the
nm; tR(1) ¼ 8.244 min (50.3%) 3b: [a]2D5 ¼ ꢀ210 (c 0.0016, CH3OH); manufacturer's protocol. Aer 5 h of transfection, the cells were
tR(2) ¼ 8.968 min (49.7%) 3a: [a]2D5 ¼ +194 (c 0.0016, CH3OH).
trypsinized and seeded onto sterilized black bottom 96-well
plates at a density of 1 ꢂ 104 cells per well. On the following day,
cells were treated with test compounds and incubated for 24 h.
Firey and Renilla luciferase activities were measured using a
NMR spectroscopy
NMR spectra were recorded at 298 K or 308 K (DMSO-d6 spectra) dual-light reporter gene assay kit (Promega) on Wallac Victor2
with a Bruker AVANCE-500 spectrometer operating at 500.13 (Perkin-Elmer, Inc., Wellesley, MA). Renilla luciferase activity
1
MHz for H, using a 5 mm single pulsed eld gradient (z-PFG) was determined to calibrate the transfection efficiency and
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cytotoxicity of chemicals. Relative STAT3 activity was calculated
by dividing the rey luciferase activity with Renilla luciferase
activity in each transfection experiment. The values of STAT3
inhibitory activity were the means of 3 experiments and the
maximum deviation from the mean was less than 10%.
5 L. Toma, G. Cignarella, D. Barlocco and F. Ronchetti, J. Med.
Chem., 1990, 33, 1591–1594.
6 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
B. Mennucci, G. A. Petersson, H. NakatsuJi, M. Caricato,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng,
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota,
R. Fukuda, J. Hasegawa, M. Ishida, T. NakaJima, Y. Honda,
O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery Jr,
J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd,
E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi,
J. Normand, K. Raghavachari, A. Rendell, J. C. Burant,
S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam,
M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev,
A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,
R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth,
P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels,
AlphaScreen-based Assay. AlphaScreen® is a bead-based
nonradioactive assay system for detecting biomolecular inter-
actions in a microtiter plate format. Binding of biological
partners brings donor and acceptor beads into close proximity
and as result, a uorescent signal between 520 and 620 nm is
produced. The AlphaScreen-based assays10 were performed in a
nal reaction volume of 25 mL of the assay buffer containing 10
mM HEPES–NaOH (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0),
0.1% NP-40, and 10 ng mLꢀ1 BSA in a 96-well microtiter plate at
25 ꢁC. Phospho-Tyr (pTyr) peptide probes used in this study
were 5-carboxyuorescein (FITC)-GpYLPQTV for STAT3, FITC-
GpYDKPHVL for STAT1, and FITC-PSpYVNVQN for Grb2.
Firstly, 75 nM of each SH2-containing protein was incubated
with the test compound for 15 min. Each protein sample was
then incubated for 90 min with 50 nM of its corresponding
FITC-pTyr peptide, and mixed with streptavidin coated donor
beads and anti-FITC acceptor beads simultaneously before
detection at 570 nm using EnVison Xcite (PerkinElmer).
¨
O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and
D. J. Fox, Gaussian 09, Revision A.02, Gaussian, Inc.,
Wallingford CT, 2009.
7 (a) P. J. Stephens, F. J. Devlin, J. R. Cheeseman, M. J. Frisch
and C. Rosini, Org. Lett., 2002, 4, 4595–4598; (b) B. Mennucci,
J. Tomasi, R. Cammi, J. R. Cheeseman, M. J. Frisch,
F. J. Devlin, S. Gabriel and P. J. Stephens, J. Phys. Chem. A,
2002, 106, 6102–6113; (c) E. Giorgio, M. RoJe, K. Tanaka,
Z. Hamersak, V. Sunjic, K. Nakanishi, C. Rosini and
N. Berova, J. Org. Chem., 2005, 70, 6557–6563; (d)
A. C. Petrovic, J. He, P. L. Polavarapu, L. S. Xiao and
D. W. Armstrong, Org. Biomol. Chem., 2005, 3, 1977–1981;
Acknowledgements
The authors acknowledge the Universities of Milan and Pavia,
and CINECA for the allocation of computer time. This study was
supported by funds from PRIN 2010-2011. B. M. Kwon was
supported by the Bio & Medical Technology Development
Program (2012M3A9C404877).
`
(e) M. Zappala, G. Postorino, N. Micale, S. Caccamese,
N. Parrinello, G. Grazioso, G. Roda, F. S. Menniti, G. De
Sarro and S. Grasso, J. Med. Chem., 2006, 49, 575–581.
8 B. A. Sherf, S. L. Navarro, R. R. Hannah and K. V. Wood,
Promega Notes Mag., 1996, 57, 2–8.
Notes and references
1 J. E. Darnell Jr, Science, 1997, 277, 1630–1635.
2 D. Masciocchi, A. Gelain, S. Villa, F. Meneghetti and
D. Barlocco, Future Med. Chem., 2011, 3, 367–397.
3 D. Masciocchi, S. Villa, F. Meneghetti, A. Pedretti, L. Legnani,
L. Toma, B.-M. Kwon, S. Nakano, A. Asai and A. Gelain,
MedChemComm, 2012, 3, 592–599.
4 D. Masciocchi, A. Gelain, F. Meneghetti, A. Pedretti,
G. Celentano, D. Barlocco, L. Legnani, L. Toma,
B.-M. Kwon, A. Asai and S. Villa, MedChemComm, 2013, 4,
1181–1188.
9 D.-S. Shin, H.-N. Kim, K. D. Shin, Y. J. Yoon, S.-J. Kim,
D. C. Han and B.-M. Kwon, Cancer Res., 2009, 69, 193–202.
10 Y. Uehara, M. Mochizuki, K. Matsuno, T. Haino and A. Asai,
Biochem. Biophys. Res. Commun., 2009, 380, 627–631.
11 (a) K. Wolinski, F. James, J. F. Hinton and P. Pulay, J. Am.
Chem. Soc., 1990, 112, 8251–8260; (b) R. Ditcheld, Mol.
Phys., 1974, 27, 789–807.
12 T. Helgaker, M. Watson and N. C. Handy, J. Chem. Phys.,
2000, 113, 9402–9409.
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