Synthesis of New Quinoxalines Containing an Oxirane Ring by the TDAE Strategy and in Vitro Evaluation in Neuroblastoma Cell Lines

Neuroblastoma is an aggressive pediatric malignancy with significant chemotherapeutic resistance. In order to obtain new compounds active on neuroblastoma cell lines, we investigated the reactivity of carbanion formed via TDAE in quinoxaline series. The new synthesized compounds were tested for their anti-proliferative activity on two neuroblastoma cell lines, and seven oxirane derivatives obtained interesting activities.


Introduction
Neuroblastoma is a neuroendocrine tumor that remains a major therapeutic challenge in pediatric oncology. Treating it requires aggressive multimodal therapy. As response rates to chemotherapy are low, surgery remains the only effective treatment. However, since many tumors have metastasized at the time of diagnosis, long-term survival rates for children with advanced disease are poor, and OPEN ACCESS undesirable side effects frequently occur. Accordingly, there is a substantial need for new therapeutic options.
In continuation of our research program centered on the design and synthesis of novel bioactive molecules [17][18][19][20][21], we decided to explore the anti-proliferative potential of quinoxaline compounds.

Chemistry
In the present study, 13 quinoxalines were evaluated against human neuroblastoma (SK-N-SH and IMR-32-SK-N-SH and IMR-32 are not acronyms but the appellations of cell lines) cell lines, 12 of which were prepared in our lab (clinical compound XK469 was purchased). Apart from molecules 8, 15, 16, all synthesized molecules bear an oxirane moiety. Compounds synthesized after molecules 3b and 4a were observed to have an interesting activity against SK-N-SH and IMR-32 cell lines. Thus, a structural homogeneous quinoxaline series was obtained, presenting a structural analogy with XK469.

Scheme 2. Synthesis of 2-(dibromomethyl)quinoxaline 2.
Oxiranes 3-4a-b resulted from the reaction of 2-(dibromomethyl)quinoxaline with aromatic aldehydes in the presence of TDAE (Scheme 3) [22].  The α-bromo carbanion formed by the action of the TDAE could react as a base with the 1H-pyrrole-2-carbaldehyde, leading to the corresponding pyrrolic anion and to the 2-bromomethyl-quinoxaline. The formation of Compound 8 could thus result from a nucleophilic substitution between these two species (Scheme 5).
To study the effect of steric hindrance, we investigated the reaction of 2-(dibromomethyl)quinoxaline 2 with two isatin derivatives, 9-10, in the presence of TDAE under classical TDAE conditions. These reactions led to a mixture of like/unlike-isomers of corresponding oxiranes 9-10a-b in good yields, as shown in Scheme 6. Diastereoisomers were separable, and their configuration was identified by NMR-analysis from the γ-left effect, as previously described [23]. Finally, the impact of the oxirane group on biological activity was studied. Thus, the reaction observed with Compound 6 was extended to other pyrrole derivatives (Scheme 7).

Scheme 7.
Reactivity of various carbonyl pyrrole in the presence of TDAE.
Results of these evaluations are summarized in Table 1. The IC50 (concentration that inhibits 50% of cell proliferation) values show clearly that the presence of an oxirane nucleus is necessary to obtain anti-proliferative activity on the two tested neuroblastoma cell lines. The reaction between the 2-(dibromomethyl)quinoxaline 2 and the pyrrole derivatives led to three compounds, 8, 15, 16, for which no activity was observed on either of the cell lines at the maximal tested dose. All oxirane derivatives displayed substantial anti-proliferative activity towards neuroblastoma cell lines. These results confirm that the oxirane group played a key role in the anti-proliferative activity [27][28][29][30].
This first study on only four couples of stereoisomers does not allow us to end on the importance of the stereochemistry on the activity. The synthesis and the evaluation of new couples of stereoisomers with an oxirane group can allow one to appreciate the importance of the stereochemistry.
This initial structure activity relationship (SAR) study will be pursued. The nature of the heterocyclic aldehyde and the use of azaheterocycles other than quinoxaline are currently under investigation.

General
Melting points were determined on a Büchi B-540 and are uncorrected. Elemental analyses were carried out on an Interscience Flash EA 1112 series (Thermo Finnigan, San Jose, CA, USA) elemental analyzer at the Spectropole, Faculté des Sciences, site de Saint-Jérôme. Both 1 H-and 13 C-NMR spectra were determined on a Bruker Avance 200 spectrometer (operating at 200 MHz for 1 H and 50 MHz for 13 C). 1 H and 13 C-NMR shifts (δ) were reported in parts per million (ppm) with respect to CDCl3 7.26 ppm for 1 H and 77.0 ppm for 13 C and DMSO-d6 2.50 for 1 H and 39.7 ppm for 13 C. Multiplicities were represented by s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). The adsorbent used for column chromatography was: silica gel 60 (Merck, Darmstadt, Germany, 230-400 mesh). Thin-layer chromatography was performed with Merck 60F-254 silica gel (0.25 mm layer thickness) in an appropriate solvent.
The general procedure for the reaction of 2-(dibromomethyl)-quinoxaline 2 with various aldehydes 3-6 or isatin and pyrrolic derivatives 9-13, using TDAE: To a two-necked flask equipped with a silica-gel drying tube and a nitrogen inlet was added, under nitrogen at -20 °C, 10 mL of anhydrous DMF solution of 2-(dibromomethyl)-quinoxaline 2 (0.45 g, 1.5 mmol) and various aldehydes 3-6 or isatin and pyrrolic derivatives 9-13, (4.5 mmol, 3 equivalents). The solution was stirred and maintained at this temperature for 30 min, and then the TDAE (0.3 g, 1.5 mmol) was added dropwise (via a syringe). A red color immediately developed with the formation of a white fine precipitate. The solution was vigorously stirred at −20 °C for 1 h and then warmed up to room temperature for 2 h. After this time, TLC analysis (dichloromethane) clearly showed that Compound 2 was totally consumed. The orange-red turbid solution was filtered and hydrolyzed with 80 mL of H2O. The aqueous solution was extracted with chloroform (3 × 40 mL), and the combined organic layers were washed with H2O (3 × 40 mL) and dried over MgSO4. Evaporation of the solvent left an orange solid as a crude product. Purification by silica gel chromatography (5/5 dichloromethane/ethyl acetate) and recrystallization from ethanol gave the corresponding derivatives.

In Vitro Biological Evaluation
Drugs: Stock solutions of quinoxaline were prepared in dimethylsulfoxide (Sigma). Stock solutions were aliquoted and stored at −20 °C. For culture and experiments in living cells, drugs were freshly diluted at appropriate concentrations in culture medium.
Growth experiments: Exponentially growing cells (37,500 cells/cm²) were seeded in 96-well plates for 24 h for SK-N-SH cells and for 72 h for IMR-32 cells and then incubated with the drugs for 72 h. The number of viable cells was estimated using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay according to our previous work [17]. After drug treatment, the medium was replaced by fresh medium containing MTT (0.5 mg/mL), and cells were incubated at 37 °C for 2 h. Then, the MTT solution was removed, and DMSO was used to dilute the formazan crystals formed in the surviving cells. Finally, absorbance was measured at 600 nm with a Multiskan (Ascent) plate reader. Inhibiting concentrations were graphically determined. IC50 is defined as the concentration that inhibits 50% of cell proliferation. At least three independent experiments (in triplicate) were performed, and data were expressed as the mean ± SD.

Conclusion
Having previously demonstrated how the TDAE strategy can be used to obtain a series of highly functionalized quinoxalinic oxiranes, we present herein an original reactivity initiated by TDAE with compounds possessing labile hydrogen. Biological evaluation of these synthesized compounds revealed promising anti-proliferative activity toward SK-N-SH and IMR-32 cell lines for most of the compounds bearing an oxirane group. Biological results showed that Compound 7 is more active than XK-469, a quinoxaline derivative involved in clinical trials, confirming that the oxirane group plays a key role in the anti-proliferative activity.
Further investigation on SAR and the mechanism of the anti-proliferative activity of these compounds will be carried out in next works. Moreover, due to promising results observed with Compound 7, it will be tested as potential anti-tumor drug on other cell lines.

Acknowledgments
This work was supported by the Centre National de la Recherche Scientifique. We express our thanks to Vincent Remusat for 1 H and 13 C NMR spectra recording.