Synthesis and Anticancer Evaluation of New 1,3,4-Oxadiazole Derivatives

In order to develop novel chemotherapeutic agents with potent anticancer activities, a series of new 2,5-diaryl/heteroaryl-1,3,4-oxadiazoles were designed and synthesized. The structures of the new compounds were established using elemental analyses, IR and NMR spectral data. The compounds were evaluated for their anticancer potential on two standardized human cell lines, HT-29 (colon adenocarcinoma) and MDA-MB-231 (breast adenocarcinoma). Cytotoxicity was measured by MTS assay, while cell cycle arrest and apoptosis assays were conducted using a flow cytometer, the results showing that the cell line MDA-MB-231 is more sensitive to the compounds’ action. The results of the predictive studies using the PASS application and the structural similarity analysis indicated STAT3 and miR-21 as the most probable pharmacological targets for the new compounds. The promising effect of compound 3e, 2-[2-(phenylsulfanylmethyl)phenyl]-5-(4-pyridyl)-1,3,4-oxadiazole, especially on the MDA-MB-231 cell line motivates future studies to improve the anticancer profile and to reduce the toxicological risks. It is worth noting that 3e produced a low toxic effect in the D. magna 24 h assay and the predictive studies on rat acute toxicity suggest a low degree of toxic risks.


Introduction
Cancer, a severe human health issue, is among the leading causes of death on a global scale, and so far chemotherapy remains a main treatment option adopted worldwide either alone or in conjunction with surgery and/or radiotherapy [1]. Despite the significant advancement in chemotherapy over recent decades, which led to major changes in the treatment of various cancers, the main obstacles to the success of the therapy have remained the development of tumor-cell resistance to various chemotherapeutic agents. Drug resistance, either existing before treatment (intrinsic) or generated after therapy (acquired), is responsible for most the relapses of cancer, one of the major causes of death of the disease [2,3].
In the attempt of finding effective anticancer agents, the strategy of using simple druggable scaffolds proved to be successful in finding many relevant lead compounds [4]. A large number of nitrogen-containing heterocyclic compounds were identified as valuable anticancer solutions [5]. Among these, oxadiazoles are receiving particular interest. Depending on the position of the nitrogen and oxygen atoms, the heterocycle may occur in the form of one of the following four different isomers: 1,2,3-, 1,2,4-, 1,2,5-, and 1,3,4-oxadiazole ( Figure 1) [6]. The greatest interest is involved with 1,3,4-oxadiazoles, as in the last years a large number of compounds with cytotoxicity for several tumor lines have been reported. The most active derivatives are more potent than the reference drugs, which proves the high anticancer potential of the 1,3,4-oxadiazole ring [7][8][9][10][11]. The 1,3,4-oxadiazole is an important scaffold in medicinal chemistry, with high versatility, giving rise to elevated structural diversity. In some cases, it acts as a bioisostere for carbonyl-containing compounds such esters, amides, and carbamates or as a flat aromatic linker to provide the appropriate molecular geometry. The stability of the oxadiazole ring in aqueous medium, and its capability to easily interact with bio-targets establishing π -π interactions or forming strong hydrogen bonds, justify the interest in the development of bioactive molecules containing this scaffold [6].

Synthesis Procedures
The compounds were designed so that the molecular weight is under 500 g/mol, and the number of hydrogen donors and acceptors comply with the Lipinski rule.
The new 1,3,4-oxadiazole derivatives were prepared by heating under reflux and magnetic stirring of aromatic carboxylic acids 1a-d with the hydrazide derivatives 2a-b in the presence of phosphorus oxychloride in 59-70% yields (Scheme 1). Scheme 1. Synthesis of the target compounds 3a-e. Reagents and conditions: (i) POCl 3 , reflux, 9 h.
The aromatic carboxylic acids 1a-c were prepared starting from thiophenol or the corresponding p-substituted thiophenols and phtalide, according to a previously reported procedure [27,28]. A synthetic procedure for the preparation of 2-[(benzenesulfonyl)methyl]benzoic acid (1d) was reported by Patra group [29], and it consists in the reaction of methyl 2-(bromomethyl)benzoate with sodium benzenesulfinate in dry dimethylformamide at room temperature, followed by the hydrolysis of the resulting ester with an aqueous solution of sodium hydroxide. The benefit of our synthesis method is the use of an alternative greener approach and the yield advantage.
The new 1,3,4-oxadiazole derivatives 3a-e were prepared by the treatment of aromatic carboxylic acids 1a-d with the hydrazide derivatives 2a-b in the presence of phosphorus oxychloride. The method was adapted based on previously described procedures [30,31]. Scheme 2 presents the atoms' numbering used for assigning the NMR signals of the new oxadiazole derivatives. In the 1 H-NMR spectra of the new compounds, the aromatic hydrogens gave signals in the range of 8.84-6.98 ppm. The methylene group CH 2 (H-12) presented a singlet in the range of 4.64-4.71 ppm for the compounds 3a-c and 3e. In the case of 3d, the S-oxidation induces a deshielding effect that results in an approximately 1 ppm higher chemical shift for the protons of the methylenic group. The methyl group presented a singlet at 2.22 ppm.
In the 13 C-NMR spectra, the two signals in the range of 164.50-161.87 ppm are produced by the two carbons in the oxadiazole ring. The carbon atoms of the benzene and the pyridine rings produce signals in the range of 149.80-120.41 ppm. The methylene group (C-12) is characterized by a signal at 36.54-38.70 ppm in the compounds 3a-c and 3e, and 59.38 ppm in compound 3d.
The IR spectra of the 3a-e compounds differ significantly from the corresponding spectra of the hydrazide derivatives 2a-b and those of the acids 1a-d.

Anticancer Evaluation
The amplification of the cell division process is responsible for the formation of tumors. Most tumor cells have disorders in the development of the cell cycle, which are associated with an exacerbated proliferative process and this is responsible for the evolution of the tumor process. In addition, the apoptotic process is inhibited in tumor cells [32,33]. In general, cytostatic treatment aims either to induce the apoptotic process of the tumor cells or to cause cell cycle blockage.
To determine the role of the studied compounds on tumor processes, studies were performed on two different types of cancer (colon and breast) using the standardized cell lines HT-29 and MDA-MB-231. The cells treated with the compounds 3a-e and the intermediates 2a-b were subjected to flow cytometry techniques in order to examine the apoptotic process and the cell cycle analysis. The apoptotic cells were determined by flow cytometry using Annexin V-FITC and PI (propidium iodide) double labeling. The live cell population, and the cells undergoing early apoptosis (Annexin+/PI−) and late apoptosis (Annexin+/PI+) were quantified [34,35]. The distribution of the cell cycle phases in the tumor cells treated for 24 h with the studied compounds was analyzed for their DNA content by flow cytometry [36,37].
Cisplatin (CisPt) is frequently used for the treatment of colon adenocarcinoma, while doxorubicin (DOX) is routinely used in the treatment of breast cancers, and was therefore chosen as a reference (CisPt for HT-29 cells, DOX for MDA-MB-231 cells).

Effects on Cell Viability
The cell viability after treatment with the compounds and reference drugs was determined using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium). The HT-29 and MDA-MB-231 tumor cells were preliminarily treated with the new compounds in concentrations from 6.25 µM up to 200 µM for 24 or 48 h in order to determine the optimal concentration. Two concentrations, 10 µM and 50 µM, were chosen to evaluate the compounds effect on the cells' viability.
The two cell lines responded differently to treatment with the tested compounds. In the HT-29 cell line, the tested compounds reduce viability in a similar way regardless of the concentration used ( Figure 3). In contrast, in the MDA-MB231 line, the cell viability is affected more when the compounds are used in a higher concentration (50 µM) and the chemical structure has a greater impact, the compound 3e having the strongest effect ( Figure 4). It is noteworthy that all the compounds analyzed have a stronger cytotoxic effect than the effect induced by CisPt or DOX. The new oxadiazole derivatives 3a-e reduced the HT-29 cells' viability with values in the range of 64.0% (3d) up to 73.2% (3c) when exposed for 24 h at 10 µM, and with values between 61.5% (3d) and 68.1% (3b) when the concentration was 50 µM. The effect of the compounds was higher when the exposure was doubled to 48 h. The cells' viability was between 50.3% (3d) and 57.7% (3e) for 10 µM, and between 39.0% (3e) and 49.4% (3c) for the 50 µM concentration. The compounds 3a-e reduced the MDA-MB-231 cells' viability with values in the range of 39.9% (3e) up to 57.2% (3c) when exposed for 24 h at 10 µM. The viability values were between 23.8% (3e) and 53.1% (3b) when the concentration was 50 µM. The cells' viability was between 50.8% (3d) and 56.9% (3e) after exposure for 48 h at 10 µM, and between 38.7% (3e) and 48.9% (3c) for the 50 µM concentration.

Effects on Cell Apoptosis
The HT-29 and MDA-MB-231 cells were treated for 24 h with the compounds 3a-e and the intermediates 2a-b, and subjected to a double Annexin/PI staining technique that allowed the detection of the apoptotic process by flow cytometry. CisPt and DOX were used as positive controls (Table 1). The treatment of the HT-29 cells for 24 h with 10 µM of the compounds 3a-e increased the total apoptosis in the range of 9.4% up to 51.2% compared to the untreated cells (control, 5.9 %). Considering the compound 3a, the introduction of a 4-chloro substituent is detrimental for the apoptotic effect (compound 3c), while the transformation of the sulfur atom into a sulfone (compound 3d) slightly reduced the effect. The exchange of the benzene ring with a pyridine (compound 3e) almost doubled the proportion of apoptotic cells.
The new oxadiazole compounds had greater effects on the MDA-MB-231 cells compared to those observed on the HT-29 cells, with the total apoptosis percent's in the range of 45.2% up to 62.7%. All the compounds had close to three-fold stronger effects than those of the positive control. The observed structure activity relationships are similar, but the impact of the structural transformation is smaller. The compound 3e determined the greatest effect in the oxadiazoles series, but it was smaller when compared with its precursor hydrazide 2b ( Figure 5).

Cell Cycle Analysis
A flow cytometry method was used to analyze the effect induced by the studied compounds on the cell cycle of the HT-29 tumor cells compared to the effect induced by CisPt, and on the cell cycle of the MDA-MB-231 tumor cells using DOX as a positive control ( Figure 6). The treatment of the HT-29 cells for 24 h with CisPt 10 µM induced a decrease in the G0/G1 phase from 54% to 21%, accompanied by an increase in the S phase of the cell cycle to 43.5% compared to the untreated cells (21.2%). The oxadiazoles 3a-d did not significantly alter the proportion of G0/G1 phase cells compared to untreated cells, while increasing the number of S phase cells. The compound 3e caused a different effect, augmenting the G0/G1 phase accompanied by a decrease in the S phase.
The flow cytometry analysis on the cell cycle of the MDA-MB-231 tumor cells showed a high percentage of G0/G1 phase (68.9%). The treatment with DOX 10 µM induced a synchronization of the S and G2+M phases, registering an increase in the S phase (42.4%) versus the untreated cells (5.1%), accompanied by an increase in the G2+M phases (43.9%) versus 26% in the untreated cells. The analyzed compounds determined the arrest of the cells in the G0/G1 phase, accompanied by a decrease in the S and G2+M phases. The compound 3e had the greatest impact of the oxadiazoles series, increasing the number of cells in G0/G1 and significantly reducing those in the S phase.

Daphnia Magna Toxicity Assay
The Daphnia magna (D. magna) bioassay results are summarized in Table 2. After 24 h of exposure, the compounds 3b-e induced at all the tested concentrations a lethality rate (L%) lower than 50%, whereas 3a induced an L% of 55% at the highest concentration. Due to the obtained results, the median lethal concentration (LC50) was calculated only for the compounds 3a, 2a and 2b. Although the LC50 for 2b was slightly lower than 2a, the 95%CI of both compounds suggest a similar biological response. After 48 h of exposure, all the newly tested compounds exhibited a significantly higher toxicity on D. magna. The LC50 value was not calculated for 3a and 3b because they induced an L% between 55 and 85% at all the concentrations. In the case of the compounds 3c-e, the calculated LC50 values are between 2.34 and 11.5 µM.

PASS Prediction
The prediction of an activity spectra for substances (PASS) is an algorithm that predicts a large panel of biological activities of a given molecule using its structure as input data, and yields a probability to be active (Pa) and a probability to be inactive (Pi) for each target [38]. Each compound's target profile was manually analyzed, the relevant oncotargets were selected, and the corresponding Pa values are presented in Table 3. The Pa values are an indication of the possibility that a compound interacts with a certain biological target, but not for the potency of the compound. The Pa values indicate the inhibition of STAT transcription factors, especially STAT3, as the most probable mechanism for the anti-proliferative effects of the compounds 3a-e. The results for the compounds 3d and 3a indicate that the (phenylsulfonyl)methyl substitution reduces the probability to inhibit STAT3 compared to the phenylthiomethyl substitution.  The target profile of the chemically similar compounds indicates the interaction with microRNA 21 (miR-21), Ras-related protein Rab-9A, glycogen synthase kinase-3 beta, and cellular tumor antigen p53, as the most probable interaction targets for the new synthesized compounds 3a-e.

Predicted Acute Rat Toxicity
For all the compounds, the predicted results fall in the applicability domain of the application. The predicted median lethal dose (LD50) of the new compounds after oral and intravenous (IV) administration on rats are presented in Table 4, and they indicate a relatively low degree of toxicity. All the new oxadiazole derivatives are predicted to be less toxic than their corresponding hydrazides synthesis precursors.

Discussion
A series of new 2,5-diaryl/heteroaryl-1,3,4-oxadiazoles were synthesized and evaluated for their anticancer potential on two standardized cell lines, HT-29 and MDA-MB-231. At 10 µM, all the compounds reduced the cell viability after 24 h of exposure, inducing apoptosis and perturbation of the cell cycle. The cell line MDA-MB-231 proved to be more sensitive to the compounds' action than HT-29.
The predictive studies using the PASS application indicated the inhibition of the STAT3 transcription factor as the most probable anticancer mechanism. Recent evidence shows that the 1,3,4-oxadiazole scaffold is frequently used in the structure of STAT3 inhibitors active against various cancer cells [39][40][41]. STAT3 is closely related to the occurrence of cancers and is an attractive therapeutic target for oncology and drug development. It acts in the regulation of many cellular events involving cell proliferation, differentiation, apoptosis and angiogenesis [42]. N-[2-(1,3,4-oxadiazolyl)]-4-quinolinecarboxamide, also known as STX-0119, is structurally similar to the 3a-e compounds and functions as a STAT3 dimerization inhibitor [43]. HJC0123 was developed based on the structure of STX-0119, but it does not contain the oxadiazole scaffold. When administered in the MDA-MB-231 cells, the compound blocked the phosphorylation of STAT3, reduced the cells' viability, promoted apoptosis, and increased the proportion of S-phase cells while reducing the number of cells in G0/G1 [44]. The effects of HJC0123 in the MDA-MB-231 cells are similar with those observed for compound 3e. In contrast to MDA-MB-231 tumor cell lines, STAT3 plays no major role in the colon carcinoma cell line HT-29 [45] and it could explain the lower effects of the new compounds on this cell line.
The structural similarity analysis indicated miR-21 as a highly probable target for the new compounds. MiR-21 is considered an oncomir because it is one of the most frequently up-regulated miRNA in a wide type of cancers. MiR-21 is overexpressed in the MDA-MB-231 cell line close to four-fold compared with the non-tumorigenic MCF-10A cell line. The knockdown of miR-21 suppressed the cell growth and proliferation of the MDA-MB-231 cells [46]. The levels of miR-21 are also significantly higher in the HT-29 cells and promote cell proliferation and migration [47].
The results of the predictive studies using the PASS application and the structural similarity analysis indicated STAT3 and miR-21 as the most probable pharmacological targets for the new compounds 3a-e, but also that these compounds may have multitarget activities. This is suggested also by the significant toxic effects registered in the D. magna 48 h assay.
The chemical diversity of the 3a-e structures, and the limited number of tested compounds, limits the development of structure activity relationships. The compound 3e presented the best anticancer profile of the series, probably due to the presence of the pyridine ring next to the oxadiazole structure. The promising effect of the compound 3e, especially on the MDA-MB-231 cell line, a triple-negative breast cancer line, motivates future studies to improve the anticancer profile and to reduce the toxicological risks. It is worth noting that 3e produced a low toxic effect in the D. magna 24 h assay and the predictive studies on rat acute toxicity suggest a low degree of toxic risks.

Analytical Procedures
The melting points (m.p.) were measured in open capillary tubes on an Electrothermal 9100 apparatus and are uncorrected. The 1 H-NMR and 13 C-NMR spectra were recorded on a Gemini 300 BB instrument (Varian, Palo Alto, CA, USA) at room temperature, operating at 300 MHz for 1 H and 75.075 MHz for 13 C. The chemical shifts were recorded as δ values in ppm units downfield to tetrametylsilane (TMS) used as internal standard, and CDCl 3 and DMSO-d6 as solvents. The coupling constants values (J) are reported in hertz (Hz) and the splitting patterns are abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet; and b, broad. The carbons not attached to any protons are presented as Cq, while those attached to a hydrogen atom are designated as CH.
The IR spectra were recorded on a FT/IR-4200 spectrometer (JASCO, Tokyo, Japan) with an ATR PRO450-S accessory at a resolution of 4 cm −1 . The elemental analyses were performed on a Perkin-Elmer 2400 Series II CHNS/O Elemental Analyzer (Shelton, CT, USA).

Synthesis Procedures
All the chemicals and reagents were purchased from commercial suppliers and used without purification, unless otherwise noted.

Synthesis of 2-[(benzenesulfonyl)methyl]benzoic acid (1d)
To a solution of 2-(phenylthiomethyl)benzoic acid (1a) (0.02 mol) in glacial acetic acid (100 mL), 20 mL 30% aqueous hydrogen peroxide was added dropwise. The mixture was heated for 2 h and then left overnight at room temperature. The reaction mixture was diluted with water and extracted with chloroform. The separated organic phase was dried over sodium sulfate and then concentrated under reduced pressure. The crude product was recrystallized from ethanol.

Cytotoxicity Assay
All assays were performed in triplicate in 96-well microtiter plates with flat bottom (Falcon), using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega), an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium)-based colorimetric assay. Briefly, 1 × 10 4 cells/wells were cultured in 100 µL for 24 h, culture supernatants were discarded, and then cells were treated for 24 h and 48 h with increasing concentrations of drugs. After the end of the incubation time, 20 µL reagent containing a) MTS, and b) phenazine ethosulfate (PES) were added in each well. PES has a high chemical stability that allows it to bind to MTS and form a stable solution. After adding the coloring solution, plates were incubated for 4 h at 37 • C, with mild agitation every 15 min. The method relies on the ability of metabolically active cells to reduce MTS, a yellow tetrazolium salt to the colored formazan that is soluble in the culture medium. The reduction in the tetrazolium compound to formazan was spectrophotometrically measured at λ = 492 nm, using a Dynex plate reader (DYNEX Technologies-MRS). The percentage of viability compared to untreated cells (considered 100% viable) was calculated based on the absorbance (Abs) values as follows: Cell viability (%) = (Abs treated cells − Abs culture medium)/(Abs untreated cells − Abs culture medium) × 100, Cell viability data were expressed as the mean values ± standard deviations (SD) of the experiments. Data were obtained in triplicates (n = 3), averaged and expressed as mean ± SD.

Apoptosis Analysis
The apoptosis assay was carried out using the Annexin V-FITC Kit and the manufacturer's protocol from BD Biosciences. The 5 × 10 5 cells/mL treated and untreated were suspended in cold binding buffer and stained simultaneously with 5 µL FITC-Annexin V (green fluorescence) and 5 µL PI in a dark at room temperature for 15 min. The percentages of apoptotic cells were determined by double staining with Annexin V-FITC/ PI. In each tube was added 400 µL of Annexin V binding buffer and the 5000 cells/sample were collected using FACSCantoII flow cytometer (Becton Dickinson-BD) and the analysis was performed using DIVA 6.2 software in order to identify early apoptosis (Annexin+/PI−), late apoptosis (Annexin+/PI+) and necrosis (Annexin−/PI+) [48].

Cell Cycle Analysis
The assay was carried out using Cycletest Plus DNA Reagent Kit and the manufacturer's protocol from BD Biosciences. Previously fixed cells (5 × 10 5 ) were washed twice in PBS and cell pellets were resuspended in PBS. The probes were kept in the dark and at 4 • C until data acquisition by flow cytometry using a FACSCantoII flow cytometer (Becton Dickinson-BD). The analysis was performed using ModFIT software in order to estimate the DNA index (DI) and progression through cell cycle phases [49].

Daphnia Magna Toxicity Assay
D. magna Straus was maintained parthenogenetically at 'Carol Davila' University (Department of Pharmaceutical Botany and Cell Biology). The culture was maintained at 25 • C, a photoperiod of 16 h/8 h light/dark cycle. Prior to the determination, young daphnids were selected according to their size and maintained for 24 h in artificial medium. The bioassay was performed on 10 daphnids/replicates in tissue culture plates with 12 wells (Greiner Bio-One) according to the protocol described in our previous studies [50,51]. For each compound, six concentrations were tested, ranging from 5 to 128 µM. The hydrazides 2a (20-411 µM) and 2b (20-394 µM) were used as positive controls, and a 1% DMSO solution as a negative control. The concentrations were selected based on the solubility and a pre-screening assay. The final volume/well was 4 mL, and the lethality was recorded at 24 and 48 h of exposure. All determinations were performed in duplicate. The 95% confidence intervals (95%CI) for LC50 values were also calculated using the least square fit method. All calculations were performed using GraphPad Prism v 5.1 software.

PASS Prediction
A virtual screening was performed using the computer program PASS (prediction of activity spectra for substances), a software product designed to evaluate the general biological potential drug-like molecules. The compounds were inputted in PASS as mol files and the results were analyzed if the Pa values were above the corresponding Pi values. The resulted biological targets were manually selected based on their anticancer treatment potential.

Structural Similarity Analysis
A similarity search was performed on the ChEMBL database for each compound 3a-e using a 50% threshold. The resulting structures were extracted together with their assayed activities on human targets [52]. The entries were filtered using DataWarrior v5.2.1 software [53] to remove compounds with inexact potency values and to merge duplicate structures into single entries with calculated average pIC50 or pEC50 values expressed as mol/L (M).

Prediction of the Compounds' Toxicity
The freely available program GUSAR was used to predict the LD50 values of the new compounds after oral and intravenous administration on rats [54].