Modeling , synthesis and NMR characterization of novel chimera compounds targeting STAT 3 †

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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. 1They are constituted by several structurally and functionally conserved domains: among them, the Src homology 2 (SH2) domain is essential for STATs activation cascade.STAT3, a member of the STATs 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. 2n previous researches focused on the discovery of new inhibitors targeting aberrant STAT3 signaling, we described the oxadiazole MD77, 3 a direct STAT3 SH2 domain inhibitor (IC 50 value 17.7 µM, by Alpha-Screen based assay) and the pyridazinone I, 4 exhibiting a good inhibitory activity (46% STAT3 inhibition at 2 µM 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 τ 1 -τ 4 more or less deviated from planarity. 3In 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-trifluoromethylphenyl moiety, 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 significantly populated, in complete agreement with that described in a previous paper and obtained with a molecular 55 mechanics approach. 5he main degree of conformational freedom in I is represented by the N2 side chain, able to assume two almost isoenergetic orientations, that, however, does not seem to exert a significant effect on the activity.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 75 calculations in order to identify new molecules endowed with enhanced activity against STAT3.Their synthesis was planned to verify if MD77 p-trifluoromethylphenyl moiety, which plays a crucial role for the binding to the target, could address the pyridazinone nucleus in proximity of the 80 dimerization interface.This approach started from the above stated consideration 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-trifluoromethylphenyl moiety of MD77 in a position 85 able to maintain its original orientation within the protein binding pocket.Here we report the synthesis of 1-3, the complete exploration of their conformational space performed through theoretical calculations, the racemate separations, the NMR 20 characterization as well as the results of the biological assays.

Chemistry
Starting from the commercially available α-phenylglutaric Reduction of the double bond by zinc in acetic acid, gave a mixture of the two diastereoisomers 7 and 8, which were not 60 separated and directly cyclized with hydrazine monohydrate to give the diastereoisomers 1 and 2 which were separated by flash chromatography.Dehydrogenation of the dihydropyridazinonic ring of 1 was carried out with sodium 3nitrobenzenesulfonate to give 3 while 2 did not react under the 65 same conditions.The relative configuration of 1 and 2 was 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.70

Computational studies
The modeling studies on the diastereoisomers 1 and 2, and on compound 3 were carried out with the Gaussian09 program 75 package using the B3LYP exchange-correlation functional at the 6-311+G(d,p) level. 6The 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 80 degrees of conformational freedom were considered, namely inversion of the central hexacyclic ring and rotation along the single bond connecting the amide carbonyl group to this ring, with energy minimization and optimization of all the possible starting geometries.After the optimization in vacuo, a further 85 optimization was performed at the same level as above using a polarizable continuum solvent model (PCM), choosing chloroform, dimethyl sulfoxide, or methanol as the solvent, for 1a, 2a and 3a, respectively.Figure 1 shows the minimum energy conformations of each compound together with the 90 corresponding relative energy and the percentage contribution to the overall population at 298 K.A very different conformational behaviour was found between the two diastereoisomers 1a and 2a.In fact, whereas in 1a the trifluoromethylphenylamido chain almost exclusively (99.9 95 %) prefers the pseudo-axial orientation, in its diastereoisomer 2a the opposite pseudo-equatorial orientation is largely preferred (96.2 %).Compound 3a closely resembles 1a in the preference for the pseudo-axial orientation, though in a lesser percentage (89.4%).100 Then, in an attempt to suggest the absolute configuration of these compounds, the specific optical rotation of each conformer of compounds 1a-3a was predicted using DFT calculations at the same level as above. 7The calculated [α] D for each conformer was weighted averaged on the basis of its 105 percentage contribution to the overall population and yielded a positive value for 2a as well as for 3a ([α] D = +375 and +387, respectively) whereas the value obtained for 1a was too low for a reliable prediction.The largely positive values predicted for 2a and 3a, compared with the largely positive 110 rotation values of the first eluted enantiomer of 2 and the second eluted enantiomer of 3 (see Experimental), allows to suggest that the former has the (6R,4aR) and the latter the (6R) configuration whereas no prediction seems possible for the enantiomers of 1.   experimental data on their 3D structure.In particular for each compound 1 H resonances were assigned and coupling constants measured.They are reported in Tables 1 and 2 together with the theoretical chemical shifts and coupling constants calculated for each optimized conformation of 1a-3a 55 and averaged on the basis of the population percentages reported in Figure 1.NOESY experiments were also performed to confirm the 1 and 2 relative configuration.In fact for the (6R,4aR)/(6S,4aS) configuration (Figure 2B), a 2.6 Å 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 95 (6R,4aS)/(6S,4aR) configuration (Figure 2A) that showed a 3.8 Å calculated distance.Actually, the comparison of the NOESY spectrum of 1 with the corresponding spectrum of 2 (see Figures S1a and S1b, ESI section) clearly showed an intense cross peak only between H-6 and H-4a of 2 whose configuration was 100 therefore ascertained to be (6R,4aR)/(6S,4aS).Consequently 1 resulted to be unambiguously the (6R,4aS)/(6S,4aR) isomer.

45
All calculations were performed using the Gaussian09 program package, 6
70and eq are referred to the most stable conformer of each compound.

Figure 2 .
Figure 2. Chemical structures of compounds 1a and 2a and their corresponding 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.25 50in the binding to this domain.Conclusion 55During our studies 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 60 identified as important for the STAT3 activity.The computational analysis on 1-3 revealed a very different conformational behavior of these compounds.In fact, while in 1 the trifluoromethylphenylamido chain almost exclusively prefers the pseudo-axial orientation, in 2 the opposite pseudo-65 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-trifluoromethylphenyl group with respect 70 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 racemate resolved.
80 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%.AlphaScreen-based Assay.AlphaScreen® is a bead-based nonradioactive assay system for detecting biomolecular 85 interactions in a microtiter plate format.Binding of biological partners brings donor and acceptor beads into close proximity and as result, a fluorescent signal between 520 and 620 nm is produced.The AlphaScreen-based assays10 were performed in a final reaction volume of 25 μL of the assay buffer containing 10 90 mM HEPES-NaOH (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0), 0.1% NP-40, and 10 ng/μL BSA in a 96-well microtiter plate at 25 °C.Phospho-Tyr (pTyr) peptide probes used in this study were 5-carboxyfluorescein (FITC)-GpYLPQTV for STAT3, FITC-GpYDKPHVL for STAT1, and FITC-95 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 100 before detection at 570 nm using EnVison Xcite (PerkinElmer).

Table 3
Dual luciferase assay results of compounds 1-3 Then, high field NMR spectroscopy and NOESY experiments 75 were performed to give experimental support to the calculations and unambiguously assign the 1 and 2 relative configuration.The vicinal J 6,5ax coupling constant of 1 and 2 (Table2) is in complete agreement with, respectively, an axial and equatorial orientation of the trifluoromethylphenylamido 80 chain.The combination of the observed J 6,5 and J 5,4a in the two distereoisomers is compatible with the assignment of the (6R,4aS)/(6S,4aR) relative configuration 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 85spectrum of 2, supports again its relative configuration, that makes these two hydrogens oriented on the same face of the molecule and relatively close (2.6 Å from the theoretical calculations) and not the diastereoisomeric (6R,4aS)/(6S,4aR) configuration, that makes them trans-oriented and more chemical shifts are reported as δ (ppm) using the solvent as internal standard.The enantiomeric separations of compounds 1 115 and 3 were performed by a chiral semi-preparative HPLC J = 4.8 and J = 4.8 Hz, CH), 2.65 (ddd, 1H, J = 17.4,J = 4.8 and J = 4.8 Hz, CH), 2.92 (ddd, 1H, J = 17.4,J = 12.0 and J = 4.8 Hz, CH), 3.99 (dd, 1H, J = 4.8 and J = 4.8 Hz, CH), 7.37 (dd, 1H, J = 7.50 and J = 1.0 Hz, ArH), 7.39 (ddd, 1H, J = 7.50, J = 7.50 8RL).Cell cultures were maintained at 37 °C under a humidified atmosphere of 5% CO 2 in an incubator.Transient transfection and dual-luciferase assays:8HCT-116 cells were seeded at a density of 10 × 10 5 cells in 100 mm 2 culture plate.65Thecells were co-transfected with pSTAT3-TA-Luc (27 μg/plate) and internal control plasmid pRL-TK (9 μg/plate) containing the Renilla luciferase gene.All plasmids used in this experiment were purchased from Promega.The transfection was carried out using TransFectin (Bio-Rad) according to the 70 manufacturer's protocol.After 5 h of transfection, the cells were trypsinized and seeded onto sterilized black bottom 96-well plates at a density of 1 × 104 cells per well.On the following day, cells were treated with test compounds and incubated for 24 h.Firefly and Renilla luciferase activities were measured using dual-light 75 reporter gene assay kit (Promega) on Wallac Victor2 (Perkin-Elmer, Inc., Wellesley, MA).Renilla luciferase activity was determined to calibrate transfection efficiency and cytotoxicity of chemicals.Relative STAT3 activity was calculated by dividing the firefly luciferase activity with Renilla luciferase activity in