Synthesis and anticancer activity of bis(2-arylimidazo[1,2-a]pyridin-3-yl) selenides and diselenides: the copper-catalyzed tandem C–H selenation of 2-arylimidazo[1,2-a]pyridine with selenium

Most heteroaryl selenides and diselenides are biologically active, with some reported to act as antioxidants and show activities that are medicinally relevant; hence, the development of efficient methods for their synthesis is an important objective. Herein, a simple method for the synthesis of selenides and diselenides bearing imidazo[1,2-a]pyridine rings and their anticancer activity are described. The double C–H selenation of imidazo[1,2-a]pyridine with Se powder was catalyzed by CuI (10 mol %) ligated with 1,10-phenanthroline (10 mol %) at 130 °C under aerobic conditions. The selenides or diselenides were prepared almost selectively using selenium powder in an appropriate quantity under otherwise identical reaction conditions. The prepared selenides and diselenides bearing two imidazo[1,2-a]pyridine rings were all novel compounds. Among the prepared diselenides and selenides that exhibited cytotoxicity against cancer cells, bis[2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl] diselenide showed an excellent anticancer activity and low cytotoxicity toward noncancer cells, suggesting that this diselenide is a potential lead compound for anticancer therapy.


Results and Discussion
We previously reported that the Cu-catalyzed C-H selenation of 2-phenylimidazo[1,2-a]pyridine (1a) with 1 equiv of Se powder in the presence of 10 mol % CuI and 1,10-phenanthroline in DMSO at 130 °C under aerobic conditions generated bis(2phenylimidazo[1,2-a]pyridin-3-yl) diselenide (Table 1, entry 1) [32]. In order to investigate the influence of the amount of Se powder used, 1a was reacted with 0.75 and 0.5 equiv of Se powder under the previously reported optimal experimental conditions. The reaction using 0.75 equiv of Se powder gave the diselenide 2a in 40% yield and the selenide 3a in 58% yield ( Table 1, entry 2), while the use of 0.5 equiv of Se powder resulted in the exclusive formation of the selenide 3a in 74% yield (Table 1, entry 3). These result showed that either the  To demonstrate the scope and limitations of the developed C-H selenation process, the reactions of various imidazopyridines 1 with 1 and 0.5 equiv of Se powder, respectively, were investigated under aerobic conditions in DMSO using the CuI (10 mol %) and 1,10-phenanthroline (10 mol %) catalytic system at 130 °C, the results of which are summarized in Figure 3 and Figure 4. Initially, we examined the syntheses of the diselenides 2 using 1 equiv of Se powder (2.0 mmol) relative to the 2-arylimidazopyridines 1b-i (2.0 mmol). The 2-phenylimidazopyridines 1b-d afforded the corresponding diselenides 2b-d bearing methoxy, methyl, and fluoro substituents at the 6-position of the respective imidazopyridine rings in good to high yield (77-88%, Figure 3). Furthermore, the 2-arylimidazopyridines 1f-h also afforded the corresponding products 2f-h in a yield of 82-89%. The compounds 1e and 1i, bearing a trifluoromethyl group, gave complex mixtures, and the expected products 2e and 2i were not obtained, which suggests that this reaction was sensitive to the electronic nature of the substituent of the imidazopyridine derivative. In addition, the selenide 3 was formed as a minor byproduct, and 2 and 3 were easily separated by silica gel column chromatography (yield of 3b-d, 3f, and 3g, respectively: 6-10%).
We next synthesized the selenides 3 using 0.5 equiv of Se powder (Figure 4). The reactions of Se powder with the imidazopyridines 1b-d, f, and h, bearing methoxy, methyl, and fluoro substituents as R 1 or R 2 , gave the corresponding selenides 3b-d, f, and h in good to excellent yield (78-90%). In contrast, the imidazopyridines bearing a trifluoromethyl group gave complex mixtures from which the expected products 3e and 3i were not obtained. The substituents R 1 and R 2 appeared to influence these reactions in the same manner as they did during the syntheses of the diselenides 2. It is noteworthy that this reaction only gave the selenide 3, with no diselenide byproduct observed.
Control experiments were carried out in order to investigate the reaction mechanism (Scheme 1). Heating the diselenide 2a under the standard conditions gave the selenide 3a in only 14% yield (reaction i, Scheme 1), which suggests that the conversion of 2a into to selenide 3a was difficult following diselenide formation. The reaction of the diselenide 2a with 1 equiv of the imidazopyridine 1a gave the selenide 3a in a high yield (reaction ii, Scheme 1). Moreover, the reaction of the selenide 3a with Se powder afforded the diselenide 2a in 62% yield (reaction iii, Scheme 1). These results show that the diselenide 2a We considered that the reaction mechanism may be similar to the C sp² -H selenation of imidazo[1,5-a]pyridines with Se powder proposed by Murai [34]. Based on this report and the above-mentioned control experiments, a plausible double selenation mechanism is shown in Scheme 2 and Scheme 3. The first step of the reaction involves the generation of the interme- The anticancer activity of the novel synthesized bis(2-arylimidazo[1,2-a]pyridin-3-yl) diselenides 2 and selenides 3 was evaluated in human cervical cancer HeLa cells ( Figure 5). At 25 µM, each compound significantly decreased the cell viability compared to the control group. When compared, each diselenide 2 was more toxic to HeLa cells than the corresponding selenide 3. Moreover, the 4-methoxyphenyl-containing diselenide 2f and the corresponding selenide 3f exhibited the highest anticancer activity among the diselenides and selenides, respectively. Thus, the 4-methoxyphenyl group appeared to enhance the anticancer activity of the bis(2-arylimidazo[1,2a]pyridin-3-yl) diselenides and selenides. Furthermore, only the compound 2f showed a higher anticancer activity than doxorubicin (DOX), a well-known anthracycline-based anticancer drug. As shown in Figure 6, 2f also exhibited cytotoxicity against U251 human glioblastoma cells and HKBMM human malignant meningioma cells. Importantly, the diselenide 2f was Scheme 1: Control reactions.

Scheme 3:
Proposed mechanism (2). more cytotoxic toward HeLa cancer cells than to human brain microvascular endothelial (HBME) cells, which are noncancerous ( Figure 7). Hence, the diselenide 2f may possibly be highly cytotoxic toward various cancer cell lines and relatively low cytotoxic against noncancer cells.

Conclusion
The Cu-catalyzed double C-H selenations of imidazo[1,2a]pyridines with Se powder afforded novel bis(2-arylimidazopyridin-3-yl) diselenides in satisfactory yields under mild reaction conditions. This reaction also produced the correspond-  HeLa cells and human brain microvascular endothelial (HBME) cells were exposed to compound 2f for 48 h. The cell viability was confirmed by MTT assays. The data are represented as mean ± SD values of four samples. Statistical significance when compared to the corresponding HeLa cell group at each concentration point: **p < 0.01.
ing selenides by simply controlling the amount of Se powder used in the reaction. Among the novel compounds synthesized, the bis[2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl] diselenide 2f exhibited an excellent anticancer activity toward various cancer cell lines and relatively low cytotoxicity toward noncancer cells, suggesting that the diselenide 2f may potentially be an anticancer therapeutic drug. The application of this reaction to other heterocycles, as well as detailed studies into the C-H selenation and anticancer mechanisms are currently underway in our group.

Supporting Information
Supporting Information File 1 Experimental and cytotoxicity assay details, compound characterization and X-ray data, NMR spectra.

Supporting Information File 2
Crystal data for 2a.