New Heterocyclic Compounds from Oxazol-5(4H)-one and 1,2,4-Triazin-6(5H)-one Classes: Synthesis, Characterization and Toxicity Evaluation

This paper describes the synthesis of new heterocycles from oxazol-5(4H)-one and 1,2,4-triazin-6(5H)-one classes containing a phenyl-/4-bromophenylsulfonylphenyl moiety. The oxazol-5(4H)-ones were obtained via condensation of 2-(4-(4-X-phenylsulfonyl)benzamido)acetic acids with benzaldehyde/4-fluorobenzaldehyde in acetic anhydride and in the presence of sodium acetate. The reaction of oxazolones with phenylhydrazine, in acetic acid and sodium acetate, yielded the corresponding 1,2,4-triazin-6(5H)-ones. The structures of the compounds were confirmed using spectral (FT-IR, 1H-NMR, 13C-NMR, MS) and elemental analysis. The toxicity of the compounds was evaluated on Daphnia magna Straus crustaceans and on the budding yeast Saccharomyces cerevisiae. The results indicate that both the heterocyclic nucleus and halogen atoms significantly influenced the toxicity against D. magna, with the oxazolones being less toxic than triazinones. The halogen-free oxazolone had the lowest toxicity, and the fluorine-containing triazinone exhibited the highest toxicity. The compounds showed low toxicity against yeast cells, apparently due to the activity of plasma membrane multidrug transporters Pdr5 and Snq2. The predictive analyses indicated an antiproliferative effect as the most probable biological action. The PASS prediction and CHEMBL similarity studies show evidence that the compounds could inhibit certain relevant oncological protein kinases. These results correlated with toxicity assays suggest that halogen-free oxazolone could be a good candidate for future anticancer investigations.


Introduction
The necessity of discovering new drugs that are more active and less toxic than those in use has boosted the synthesis of an increasing number of heterocyclic compounds. More than 85% of biologically active compounds possess a heterocyclic structure, and the vast majority of these contain nitrogen as a heteroatom [1,2]. The chemistry of heterocycles represents the most complex branch of organic and medicinal chemistry. Many aspects related to the structure, reactivity, synthesis and physico-chemical properties of heterocycles have captivated researchers, with an ultimate focus on revealing their biological activities and applications in various fields, including medicine, agriculture and industry [3]. Most of the known heterocycles are of natural origin, serving as a backbone in biologically active natural products used as traditional medication, or are synthetic compounds, many of them being subunits of active substances in the composition of various market drugs [2]. Among them, five-membered heterocyclic compounds from the 1,3-oxazole class have earned a distinct place in medicinal and pharmaceutical chemistry as a promising class in drug design. Oxazole is recognized as an efficient pharmacophore, being present in the structure of the active substances of some drugs, such as mubritinib (tyrosine kinase inhibitor) with antitumoral activity, oxaprozine (COX-2 inhibitor) or ditazole (platelet aggregation inhibitor) with anti-inflammatory activity, sulfamoxole (antibacterial) and aleglitazar (antidiabetic) [4][5][6] ( Figure 1). Oxazolones in particular, depending on the position of the carbonyl group and the double bond, occur in five isomeric forms, the most important and best-studied being oxazol-5(4H)-ones. These compounds, also known as azlactones, are classified as saturated or unsaturated, according to the type of exocyclic bond linked to the carbon from four position of this core [7]. Unsaturated oxazolones are important synthons for the synthesis of different compounds with vast biological potential, including amino acids, peptides, amides [8][9][10][11][12] and five-or six-membered heterocycles (imidazolones, tiazolones, triazinones, etc.) [7][8][9]12]. A great number of 4-aryliden-oxazol-5(4H)-ones have a broad spectrum of biological activities that include antitumor [13,14], antibacterial [13,[15][16][17], antifungal [13,[15][16][17], anti-inflammatory [18,19], antioxidant [10,16,20] and antidiabetic [21] properties.
Diaryl sulfone is another potent pharmacofore present in the structure of various bioactive compounds with antitumoral [31], antibacterial [32,33], antifungal [33], antioxidant [33] and antiviral [34] activity. Moreover, there are drugs with a sulfonyl group used in therapy; for example, dapsone is a commercially available antibacterial drug used to treat leprosy and various other infectious and chronic inflammatory diseases, also having antitumoral activity [31,35].
Diaryl sulfone is another potent pharmacofore present in the structure of various bioactive compounds with antitumoral [31], antibacterial [32,33], antifungal [33], antioxidant [33] and antiviral [34] activity. Moreover, there are drugs with a sulfonyl group used in therapy; for example, dapsone is a commercially available antibacterial drug used to treat leprosy and various other infectious and chronic inflammatory diseases, also having antitumoral activity [31,35].

Chemistry
Unsaturated azlactones can be obtain using synthetic procedures such as Erlenmeyer reaction, Bergmann synthesis, the reaction of ketoacids with primary amides or other catalytic methods under different conditions, the most facile, convenient and efficient being the first indicated method [43].
The new oxazol-5(4H)-ones 3a,b,d were synthesized according to the Erlenmeyer method [43], via the cyclocondensation of the 2-(4-(4-X-phenylsulfonyl)benzamido)acetic acids 1 [44,45] with benzaldehyde or 4-fluorobenzaldehyde 2, under reflux, using acetic anhydride as a dehydrating agent and in the presence of sodium acetate, with 38-80% yields. The derivative 3c has already been reported [38]. The new 1,2,4-triazin-6(5H)-ones 4a-d were obtained via the condensation of the oxazolones 3a-d with phenylhydrazine in acetic acid and in the presence of sodium acetate, under reflux, with 42-84% yields. The synthesis of key intermediates acids 1 was accomplished as previously described [44,45] via the N-acylation of glycine with 4-(4-X-phenylsulfonyl)benzoyl chlorides [46] that were prepared beginning with the alkylation of benzene or bromobenzene with p-toluenesulfonyl chloride, followed by oxidation of corresponding diaryl sulfones, and finally, the reaction of 4-(4-X-phenylsulfonyl)benzoic acids with thionyl chloride (Scheme 1). The structures of the newly synthesized compounds were established based on the recorded spectral data (IR, 1 H-NMR, 13  acids 1 [44,45] with benzaldehyde or 4-fluorobenzaldehyde 2, under reflux, using acetic anhydride as a dehydrating agent and in the presence of sodium acetate, with 38-80% yields. The derivative 3c has already been reported [38]. The new 1,2,4-triazin-6(5H)-ones 4a-d were obtained via the condensation of the oxazolones 3a-d with phenylhydrazine in acetic acid and in the presence of sodium acetate, under reflux, with 42-84% yields. The synthesis of key intermediates acids 1 was accomplished as previously described [44,45] via the N-acylation of glycine with 4-(4-X-phenylsulfonyl)benzoyl chlorides [46] that were prepared beginning with the alkylation of benzene or bromobenzene with p-toluenesulfonyl chloride, followed by oxidation of corresponding diaryl sulfones, and finally, the reaction of 4-(4-X-phenylsulfonyl)benzoic acids with thionyl chloride (Scheme 1). The structures of the newly synthesized compounds were established based on the recorded spectral data (IR, 1 H-NMR, 13  The IR spectra of new oxazolones 3 confirmed the cyclocondensation of N-acylated glycine derivatives 1 with aldehydes through the disappearance of the NH absorption band from 3416 cm −1 (X = H) and 3341 cm −1 (X = Br) of these precursors [44,45]. The stretching vibration of the C=O lactone group from the oxazolones 3 is highlighted by a characteristic double absorption due to Fermi resonance, between 1771 and 1798 cm −1 [10,17,47]. In the IR spectra of new compounds 4a-d, the appearance of a new absorption band in the range 3231-3331 cm −1 , characteristic of stretching vibration of the NH group from the triazinone ring, confirms that the reaction of oxazolones 3a-d with phenylhydrazine took place. Also, the absorption band from 1709 to 1720 cm −1 is due to stretching vibration of the C=O group from the triazinone ring. In the 1 H-NMR spectra of the new compounds 3, the most important proof that the condensation reaction occurred was the disappearance of the triplet signal due to the NH proton from precursors 1 (9.09 ppm when X = H [45] and 8.20 ppm when X = Br [44]) and the presence of a new singlet characteristic of the proton from the =CH group, with a chemical shift δ in the range of 7.43-7.46 ppm. In the case of compounds 4, the new singlet signal characteristic of the NH proton from the triazinone ring appeared at δ = 8.98-9.00 ppm, and the =CH proton from the phenylhydrazine moiety resonated between 7.33 and 7.36 ppm as a singlet signal. The oxazolone ring is confirmed in the 13 C-NMR spectra by the signals of the C-2 (161.5-161.7 ppm), C-4 (132.3-135.0 ppm) and C-5 (166.3-166.4 ppm) atoms. The signals of the C-3 and C-5 atoms from triazinone core are highlighted at δ values between 159.5 and 159.7 ppm and 133.7 and 135.7 ppm, respectively. The =CH carbon signal from oxazolones 3a-d appeared at δ = 131.2-132.6 ppm and in triazinones 4a-d in region 128.47-129.85 ppm. Also, the C=O carbon from triazinones resonated in the range 168.7-168.8 ppm. The results of the toxicity evaluation of oxazolones 3a-d and triazinones 4a-d on Daphnia magna are presented in Table 1 and Figure 2. After 24 h of exposure, the tested compounds induced a lethality of less than 30%, and therefore the LC 50 could not be calculated. After 48 h of exposure, triazinones 4a-d induced a higher toxicity compared to oxazolones 3a-d. Of the oxazolone derivatives, compound 3a induced the lowest toxicity.
In the case of the other oxazolones, due to having a fluorine atom on the benzylidene fragment or a bromine atom on the phenylsulfonylphenyl moiety, or both halogens, the toxicity was increased. Among all tested compounds, triazinone 4b, containing a fluorine atom on the arylidene fragment grafted to the 5 position of the heterocyclic core, was the most toxic. Triazinone derivatives 4c and 4a also induced a high toxicity, followed by 4d, which showed a moderate to high action, suggesting a major effect of the triazinone nucleus, which was probably influenced by the halogen atom.

Saccharomyces cerevisiae Toxicity Assay
The toxicity against the S. cerevisiae cells was evaluated by monitoring the cell s proliferation when exposed to compounds 3a-d and 4a-d. The yeast cells were incubated for 24 h in rich YPD medium supplemented or 0.1 mM and 1 mM solutions of compounds

Saccharomyces cerevisiae Toxicity Assay
The toxicity against the S. cerevisiae cells was evaluated by monitoring the cell's proliferation when exposed to compounds 3a-d and 4a-d. The yeast cells were incubated for 24 h in rich YPD medium supplemented or 0.1 mM and 1 mM solutions of compounds 3a-d or 4a-d. Cell growth in the presence of each compound was calculated relative to the cell growth in the absence of any compound, but with an equivalent DMSO concentration. These two concentrations were set as the limits of the concentration range, since it was noted that compounds 3a-d and 4a-d were practically non-toxic at concentrations of less than 0.1 mM, while concentrations higher than 1 mM were cumbersome, causing precipitation in the incubation medium. The effect of compounds 3a-d and 4a-d (at 0.1 mM and 1 mM) on the growth of S. cerevisiae is presented in Figure 3.  As seen in Figure 3, the compounds exhibited low toxicity at a 0.1 mM concentration, and the growth of the yeast cells exposed to 0.1 mM 3a-d or 4a-d was not significantly different compared to the control. On the other hand, at 1 mM, the highest toxicity was noted for triazinone 4c, followed by its corresponding precursor, oxazolone 3c. At 1 mM, oxazolone 3c reduced the growth of the yeast by 57.4% and triazinone 4c reduced the growth by 63.5%, suggesting that introducing a bromine atom in the structure of these compounds but also the presence of this six-membered core (4c) might increase the toxicity.
The least toxic compounds at either 0.1 mM (3d-94.2%, 4d-99.6%) or 1 mM (3d-89.3%, 4d-89.7%) seemed to be the pair 3d-4d, the oxazolone and triazinone that present both a bromine and a fluorine atom in their structures. In the presence of these compounds, the growth of the S. cerevisiae was practically unhindered (Figure 3). This was rather surprising, indicating that the increased tolerance to compounds 3a/4a, 3b/4b or 3d/4d may be caused by active extrusion of the compounds from the cytosol via ATPdependent multidrug transporters. As the apparent tolerance could be the result of plei- As seen in Figure 3, the compounds exhibited low toxicity at a 0.1 mM concentration, and the growth of the yeast cells exposed to 0.1 mM 3a-d or 4a-d was not significantly different compared to the control. On the other hand, at 1 mM, the highest toxicity was noted for triazinone 4c, followed by its corresponding precursor, oxazolone 3c. At 1 mM, oxazolone 3c reduced the growth of the yeast by 57.4% and triazinone 4c reduced the growth by 63.5%, suggesting that introducing a bromine atom in the structure of these compounds but also the presence of this six-membered core (4c) might increase the toxicity.
The least toxic compounds at either 0.1 mM (3d-94.2%, 4d-99.6%) or 1 mM (3d-89.3%, 4d-89.7%) seemed to be the pair 3d-4d, the oxazolone and triazinone that present both a bromine and a fluorine atom in their structures. In the presence of these compounds, the growth of the S. cerevisiae was practically unhindered (Figure 3). This was rather surprising, indicating that the increased tolerance to compounds 3a/4a, 3b/4b or 3d/4d may be caused by active extrusion of the compounds from the cytosol via ATP-dependent multidrug transporters. As the apparent tolerance could be the result of pleiotropic drug resistance, the growth of yeast cells defective in plasma membrane multidrug transporters Pdr5 and Snq2 [41,42] exposed to various concentrations of compounds 3a-d and 4a-d (Figure 4) was determined.
It was noticed that both pdr5∆ and snq2∆ knockout mutants were more sensitive to compounds 3a/4a and 3b/4b and significantly more sensitive to 3d/4d (Figure 4) than the wild type, suggesting that these compounds may be extruded from yeast cytosol by both Pdr5 and Snq2. In contrast, the sensitivity of yeast cells to 3c/4c was not significantly altered by PDR5 or SNQ2 deletion (Figure 4). These observations suggest that either 3c/4c are not recognized by Pdr5/Snq2, or the compounds act as inhibitors of Pdr5/Snq2 activity. Pdr5 and Snq2 play an important role in the efflux of xenobiotics, as their spectrum contains a wide variety of compounds, including anticancer drugs [42].

Prediction of the Molecular Mechanism of Action
The results of the PASS analysis for compounds were manually analyzed, and the relevant oncotargets are presented with the predicted Pa values in Table 2.
The Pa values are not an indicator of a compound's potency but the probability that the compound will interact with a certain biological target or produce a specific effect. The Pa values indicate the inhibition of certain protein kinases as the major antiproliferative mechanism, especially the platelet-derived growth factor receptor kinase (PDGF-R) and focal adhesion kinase 2 (FAK2). The transformation of the oxazol-5(4H)-one fragment in the corresponding 1,2,4-triazin-6(5H)-one scaffold reduces the probability of FAK2 inhibition, but has little effect on the PDGF-R inhibitory potential.
The similarity search on the ChEMBL database returned a number of 61 structures for the compounds 3a-d and only 2 results for the related 4a-d structures. The highest degree of structural similarity (76.19%) was registered for the pair 3b and CHEMBL1972440 (4-[(4-fluorophenyl)methylene]-2-phenyloxazol-5(4H)-one). The IC 50 value represents the concentration expressed as a mol/L of each compound causing a 50% decrease in cell proliferation, and pIC 50 represents the corresponding negative log10 value. Of the 61 similar compounds found, 20 were tested on the NCI60 cell assay. The pIC 50 values for the 60 cells in the NCI database are graphically presented, with the best similarities being found for the compounds 3a-d ( Figure 5).

Prediction of the Molecular Mechanism of Action
The results of the PASS analysis for compounds were manually analyzed, and the relevant oncotargets are presented with the predicted Pa values in Table 2. degree of structural similarity (76.19%) was registered for the pair 3b and CHEMBL1972440 (4-[(4-fluorophenyl)methylene]-2-phenyloxazol-5(4H)-one). The IC50 value represents the concentration expressed as a mol/L of each compound causing a 50% decrease in cell proliferation, and pIC50 represents the corresponding negative log10 value. Of the 61 similar compounds found, 20 were tested on the NCI60 cell assay. The pIC50 values for the 60 cells in the NCI database are graphically presented, with the best similarities being found for the compounds 3a-d ( Figure 5).  Four compounds, CHEMBL1972440, CHEMBL1994121, CHEMBL1089606 and CHEMBL 1988306, share a common 4-(phenyl)methylene-2-phenyloxazol-5(4H)-one scaffold and average antiproliferative effects. The best anticancer profile was observed for CHEMBL1089606, indicating that the substitution on the aromatic rings with halogens is unfavorable. This result is similar to the results of the PASS prediction, which suggest compound 3a has a better potential to inhibit protein kinases.

General Information
All reagents and solvents were purchased commercially with high purity from Sigma-Aldrich or Merck. The melting points (m.p.) were determined using a Boëtius hot-plate microscope (VEB Wägetechnik Rapido, PHMK 81/3026, Radebeul, Germany) and are uncorrected. The IR spectra were registered on a Vertex 70 spectrometer (Bruker Optik GmbH, Ettlingen, Germany) in KBr pellets. The 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz) spectra were recorded on a Gemini 300 BB spectrometer (Varian, Inc., Palo Alto, CA, USA) in deuterated DMSO-d 6 solvent at room temperature. The values of chemical shifts (δ) in parts per million (ppm) are reported relative to tetramethylsilane (TMS) as the standard reference, and the coupling constants (J) are provided in Hz. The multiplicity of signals is abbreviated as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet; b, broad signal. The mass spectra of 3a, 3d and 4a-c were registered on a triple-quadrupole Varian 1200 LC/MS/MS mass spectrometer (Varian, Inc. Walnut Creek, CA, USA) with an ESI (electrospray ionisation) or APCI (atmospheric pressure chemical ionization) interface. The mass spectrum for 4d was acquired with the 8060NX triple-quadrupole mass spectrometer, with pump LC 40 D X3 automatic injector SIL 40 C X3 fitted with an ESI source (Shimadzu, Kyoto, Japan). Solutions of oxazolones 3 or triazinones 4 in chloroform (0.5 mg /mL) were prepared and diluted 10 times with methanol (1% formic acid for 3 and 1% ammonium carbonate for 4). The sample solutions of compounds 3 were ionized positively, and those of 4 were ionized negatively by passing them through the ESI/APCI interface with positive or negative ionization using a solution injection system with a loop mounted on a Reodyne 7725 valve and the Varian Prostar 240 SDM pump, operating at a flow rate of 50 µL/min. The protonated molecular ions of oxazolones or negative ions of triazinones were fragmented into the argon collision cell at 1.5 mTorr. The elemental analysis was carried out on a Perkin-Elmer 2400 Series II CHNS/O Elemental Analyzer (Waltham, MA, USA).

Cell Growth Assessment
Yeast pre-cultures left overnight in rich YPD medium were inoculated in fresh SC medium at a density of 2 × 10 5 cells/mL, and incubated afterwards for 2 h under shaking (200 rpm, 30 • C) in a multi-amplitude orbital constant temperature shaking incubator (Shanghai ZHICHENG Analytical Instruments Manufacturing Co., Ltd., Shanghai, China) before the solutions of the compounds 3a-d and 4a-d were added to the specified concentrations.
The growth of the yeast was determined after 24 h of exposure to the oxazolones and triazinones by measuring the turbidity of the cellular suspensions at the wavelength of 600 nm [53]. The turbidity was recorded using a plate reader equipped with a thermostat and a shaker (Varioskan, Thermo Fisher Scientific, Vantaa, Finland). The growth of the yeast cells in the presence of each tested compound was calculated relative to the cell growth in the absence of the compound, in a medium containing the equivalent concentration of dimethyl sulfoxide.

Prediction of the Molecular Mechanism of Action
The SMILES codes for the compounds 3a-d and 4a-d were introduced in the PASS (Prediction of Activity Spectra for Substances) application in order to evaluate the potential to interact with a large collection of biological relevant molecules. The output results consist of an array of pair probabilities, Pa and Pi. Pa represents the probability of the compound to interact (Pa) with a specific target, while Pi is the probability of the negative outcome (Pi) [54].
For each compound 3a-d and 4a-d, a similarity search was performed on the ChEMBL database using a 50% threshold [49,55]. The output structures were extracted together with their antiproliferative data on the NCI cell panel. The collected data were filtered using DataWarrior v5.2.1 software [56].

Conclusions
New compounds from oxazol-5(4H)-one and 1,2,4-triazin-6(5H)-one classes incorporating a diaryl sulfone moiety were synthesized and characterized, and their toxicity was evaluated on D. magna crustaceans and S. cerevisiae yeast. The new unsaturated oxazolones were prepared via cyclocondensation of some 2-(4-(4-X-phenylsulfonyl)benzamido)acetic acid intermediates with aromatic aldehydes. The new triazinone derivatives were synthesized from their oxazolone precursors via condensation with phenylhydrazine. The structures of the new synthesized compounds were established using IR, 1 H-, 13 C-NMR, mass spectral data and elemental analysis. Both the heterocyclic nucleus and the halogen atoms significantly influenced the toxicity against D. magna. The oxazolone derivatives were less toxic compared with the compounds belonging to triazinone series. The results of toxicity screening against S. cerevisiae indicate that the action of the compounds is considerably hindered by the activity of the MDR transporters Pdr5 and Snq2. The predictive studies indicated that the new compounds could inhibit cancer cells proliferation by targeting certain protein kinases, especially PDGF-R and FAK2. The results suggest that compound 3a has the best potential to inhibit oncologic protein kinases, and an average antiproliferative effect with pIC 50 values between 4 and 5. The same derivative had the lowest toxicity in the D. magna assay, thus being a good candidate for future anticancer investigations. All the results obtained in the investigation of the synthesized compounds require further study for the elucidation of the mechanisms implicated.  Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.