Selenium Dioxide–Mediated Synthesis of Fused 1,2,4-Triazoles as Cytotoxic Agents

Abstract A series of fused 1,2,4-triazoles has been prepared by oxidative intramolecular cyclization of heterocyclic hydrazones with selenium dioxide. General applicability of this practical protocol was confirmed by the synthesis of moderate to good yields of 1,2,4-triazolo[4,3-a]pyridines, 1,2,4-triazolo[4,3-a]pyrimidines, 1,2,4-triazolo[4,3-a]pyramidines, and 1,2,4-triazolo-[4,3-a]quinoxa lines. All compounds were tested in vitro for their cytotoxic activity against HCT-116, A549, and Colo-205 cell lines. Two compounds, 3-(4-methoxyphenyl)-7-methyl-[1,2,4]triazolo[4,3-a]pyridine and 1-(4-methoxyphenyl)-[1,2,4]triazolo[4,3-a]quinoxaline, showed potent antiproliferative activity against the three cell lines. GRAPHICAL ABSTRACT


RESULTS AND DISCUSSION
The precursors, hydrazones, were obtained by treating the corresponding hydrazino heterocycles with aldehydes.
To achieve a generally accepted oxidizing agent for the cyclization of hydrazine, a series of additives was examined for efficiency in the heterocyclization of hydrazone (1f). The results listed in Table 1 indicate that the SeO 2 is more effective than others, although greater reaction temperature was required. Compared with Scheme 1. Synthesis of 1,2,4-triazoles 5-8. other additives, the use of SeO 2 as a heterogeneous oxidant in this reaction has advantage of good yield, simple workup, and low cost.
The target compounds were evaluated for cytotoxic activity against three human cancer cell lines using the sulforhodamine B (SRB) assay. [16] 5-Fluorouracil (5-FU), which has a broad spectrum of anticancer activity, was used as reference drug. The three cell lines are human colon cancer HCT-116 and Colo-205 cell lines and non-small-cell lung carcinoma A549 cell line. The IC 50 values of all targeted compounds are assessed and summarized in Table 3.
As shown in Table 3, most compounds were able to inhibit the growth of the three cancer cells with IC 50 values between 2 and 70 µM. Among them, compounds 5a, 5b, and 8a (R ¼ alkyl groups) were found to be inactive against all tumor cell lines (IC 50 > 100 lM). The cytotoxic activities of 4-methoxylphenyl analogs (5f, 6a and 8b) was superior to those of other substituted-phenyl analogs. The antiproliferative activities of 1,2,4-triazolo[4,3-a]quinoxalines seemed to be stronger than those of other triazoles. In the whole series, compound 8b showed significant growth  inhibition with IC 50 values of 2.36 and 5.76 mM against HCT-116 and A549 cell lines respectively, which is almost equipotent to 5-fluorouracil.

CONCLUSIONS
In conclusion, we have developed a practical and efficient protocol for the synthesis of fused 1,2,4-triazoles by oxidation of heterocyclic substituted hydrazones using SeO 2 as oxidizing agent. The presence of several functionalities in the substrate is tolerated and almost does not affect the yield of the desired product. Most of the target compounds displayed potent antitumor activity. Among them, compound 8b exhibited significant growth inhibition with IC 50 values of 2.36 and 5.76 mM against HCT-116 and A549 cells respectively, which is almost equipotent to 5-fluorouracil.

EXPERIMENTAL
All the reagents and solvents for the synthesis and analysis were commercially available and used directly. Melting points were recorded on a YRT-3 melting-point apparatus and are uncorrected. 1 H NMR and 13 C NMR spectra were measured using a Bruker DRX 400 instrument [400 MHz for 1 H NMR and 100 MHz for the 13 C NMR (proton decoupled)] using dimethylsulfoxide (DMSO-d 6 ) as solvent and tetramethylsilane (TMS) as internal standard, chemical shifts (d scale) are reported in parts per million (ppm). Data are reported as follows: chemical shift in ppm (d), multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, brs ¼ broad singlet, m ¼ multiplet), coupling constant (Hz), integration. Mass spectra analyses of all the compounds were performed on a high-resolution q-TOF.

Typical Procedure for the Synthesis of Hydrazones (1-4)
Hydrazones 4b and 4c were prepared according to the literature procedure. [17] The new hydrazones are prepared as follows: The hydrazine (15 mmol) was dissolved in boiling ethanol (20 ml) and the aldehyde (15 mmol) dissolved in ethanol (20 ml) was added dropwise. After that, the solution was stirred and heated under reflux for 1 h. The formed hydrazone was filtered from the cooled solution and used in the next reaction without any purification.

SYNTHESIS OF FUSED 1,2,4-TRIAZOLES
and monitored by thin-layer chromatography (TLC). After the completion of the reaction, the reaction mixture was filtered, and the filtrate was evaporated in vacuum. The crude product was purified by recrystallization or by flash column chromatography to give the desired product. 3-(4-Methoxyphenyl)-7-methyl- [1,2,4]

In Vitro Antiproliferative Activity Assays
The antiproliferative activity of the compounds was tested on HCT-116, A549, and Colo-205 cells. Cells were seeded in 96-well plates at a density of 5000 cells=well and incubated for 24 h. The cells were then incubated with serially diluted compounds for another 48 h. The final number of cells per well was assessed using the sulforhodamine B dye (SRB, Sigma-Aldrich) following the standard procedure, [16] and absorbance was measured using a microplate reader (SpectraMax M5) at a wavelength of 515 nm. The results were expressed as IC 50 calculated using the Easy-fit software.

SUPPLEMENTAL MATERIAL
Experimental procedures, 1 H NMR and 13 C NMR spectra data, and references for all the products for this article can be accessed on the publisher's website.