Synthesis of novel oxadiazole derivatives and their cytotoxic activity against various cancer cell lines

Caffeic acid (CA), ferulic acid (FA) and caffeic acid phenethyl ester (CAPE) have a broad anticancer effect on various cell lines. In this study, nine ferulic and caffeic acid-based 1,2,4 and 1,3,4 oxadiazole molecular hybrids were synthesized and their cytotoxic activity was evaluated mainly against Glioblastoma (GBM) cell lines. Compounds 1 and 5 exhibited the highest inhibitory activity against three different GBM cell lines (LN229, T98G, and U87), without toxicity to healthy human mesenchymal stem cells (hMSC). In addition, their cytotoxicity was also evaluated against three additional cancer cell lines and more inhibitory results were found than GBM cell lines. The IC50 values of compound 5 in U87, T98G, LN229, SKOV3, MCF7, and A549 cells were determined as 35.1, 34.4, 37.9, 14.2, 30.9, and 18.3 μM. In the light of biological activity studies, the developed compounds have a high potential to lead studies for the development of new drug candidates for the treatment of cancer.

60. Melting points were taken in open capillary tubes using a Stuart SMP30. High resolution mass spectrum (HRMS) was measured using Thermo ORBITRAP Q-EXACTIVE instrument. The 1 H and 13 C (APT) NMR spectra were measured in CDCl 3 , CD 3 OD, or DMSO-d 6 on a Varian NMR 500 MHz NMR spectrophotometer.

Analysis of cell viability
Cells were seeded in 96-well black plates and incubated for 24 h at 37 °C in 5% CO 2 . The culture medium was removed and the cells were treated for 48 h in three different wells for each concentration (1, 10, 25, 50, 100, 250 µM) of CA, CAPE, and novel oxadiazole derivatives. Cell Titer-Glo reagent was added to each well after 48 h of treatment, and samples were analyzed on Spectramax (SpectraMax i3x Multi-Mode Detection Platform). Results were standardized relative to cell controls treated with the highest dose (0.1%) of the compound's solvent, dimethyl sulfoxide (DMSO) (Santa Cruz). IC 50 values were determined using GraphPad 8.0.2.

Statistical analysis
Experiments were carried out in three sets, each with its own set of results, which were expressed as mean standard error. All statistical comparisons were made using the Student's t-test, which claimed equal variance. At *p 0.05 and **p 0.01, the differences were declared statistically significant. The data was expressed as a standard error of the mean (SEM).

Chemistry
A recent study published by Tripathi et al., showed that the similar hybrid molecules can be effective not only on cancer cells, also used against Alzheimer's disease (AD) [28]. The inhibition of BACE-1 enzyme with our compound 8 was reported here as a therapeutic approach to treat AD. In our study, the cytotoxic activity of same compound and additional some new oxadiazoles were evaluated as well. In view of this point, some novel 1,2,4-and 1,3,4-oxadiazole analogues (1)(2)(3)(4)(5)(6)(7)(8)(9) were prepared by one-pot reactions (Figure 2). The 3,5-disubstituted-1,2,4-oxadiazoles (1-3) were obtained by the reaction of caffeic/ferulic acid and the corresponding oxime in the presence of EDC and HOBt. Since we used commercially available trans isomers of caffeic/ferulic acids as starting material, only same isomer was obtained at the end of our method. Compound 1 was synthesized for cytotoxic activity comparison with our previous work [27]. To clarify, the effect of the -CF 3 group on phenyl moiety, compounds 2 and 3 were prepared and compared their activity with compound 1. The 2,5-disubstituted-1,3,4-oxadiazoles (4-9) were synthesized to investigate the effect of 1,3,4-oxadiazole ring. These compounds were obtained by the reaction of hydrazide and caffeic/ferulic acid with POCl 3 . The synthesis of the compounds was achieved in 50%-68% yield. Following the synthesis, the crude mixtures were purified by column chromatography and PLC methods using silica gel. All compounds were characterized via 1 H NMR and 13 C NMR (APT). Furthermore, calculated and measured m/z values of the compounds were also found compatible in HRMS analysis.

Biological
Anticancer activity of the synthesized novel oxadiazole derivatives were evaluated against on U87, T98G, and LN229 GBM cell lines. The various concentrations of the derivatives were used to determine IC 50 values (Table 1)     IC 50 values of compound 1 was determined as 60.3, 39.2, and 80.4 µM in U87, T98G, and LN229 cells, respectively. To clarify the effect of -CF 3 group on the inhibitory activity, the result of compound 3 was compared with compound 1, it was concluded that this group did not increase the activity positively.
In addition, compound 5, possessing 1,3,4-oxadiazole ring was found to have the highest inhibitory activity in comparison to the all other oxadiazoles and reference molecules CA and CAPE. The IC 50 values of compound 5 in U87, T98G, and LN229 cells were determined as 35.1, 34.4, and 37.9 µM, respectively (Table 1). To examine the effects of furyl and phenyl (R 3 ) rings on inhibitory activity, compounds 4 and 5 were prepared and compound 5 with furyl ring showed better inhibitory activity in GBM cell lines. Compounds 6-9 have no significant inhibitory activity on the GBM cell lines. When we compare the inhibitory activity of the compounds 4-5 with compounds 6-9, it was clear that the dihydroxy group of the 1,3,4-oxadiazoles in the phenylpropanoid structure significantly increased the activity.
Cell viability analysis was performed in SKOV3 (ovarian cancer), MCF7 (breast cancer), and A549 (lung cancer) cells for compounds 1 and 5, compound 5 showed higher cytotoxicity than references CA and CAPE in GBM cells lines. The IC 50 values are listed in Table 2.
In comparasion to GBM cell lines, SKOV3, MCF7, and A549 cells showed higher sensitivity towards compounds 5 and 1. The IC 50 values of compound 5 were determined as 14.2, 30.9, and 18.3 µM in SKOV3, MCF7, and A549 cells, respectively. Based on these results, compound 5 appears to be more active than both compound 1 and references CAPE and CA in these three different cancer cells.
In addition, cell viability assay was performed on healthy hMSC in the range of 1-50 µM values to examine the effects of compound 5 and 1 and references CAPE and CA. When the hMSC cells incubated with compounds 5, 1 and references CAPE, CA with in the 50 µM (highest concentration) values for 48 h, cell viability was found 94.59%, 57.62% and 16.89%, 19.22%, respectively ( Figure 4).

Conclusion
Novel oxadiazole derivatives were synthesized and evaluated for their anticancer activities in different cancer cells. The most promising results were obtained with compound 1 and 5, carrying 1,2,4-oxadiazole and 1,3,4-oxadiazole moieties, respectively. Compound 5 showed similar activity (35 ± 2 µM) aganist all GBM cell lines. In addition, compounds 1 and 5 significantly inhibit cell proliferation at low concentrations in different cancer cell lines, such as ovarian, breast, and lung. Moreover, the active compounds did not show any toxicity towards the selected nonmalignant cell lines. The results indicate that it is possible to synthesize various derivatives which could be used in further studies to investigate different pathways in cancer.