Synthesis, Characterization and Cytotoxicity Evaluation of New Compounds from Oxazol-5(4H)-Ones and 1,2,4-Triazin-6(5H)-Ones Classes

ELENA VALENTINA ROSCA1, THEODORA VENERA APOSTOL1*, CONSTANTIN DRAGHICI2, OCTAVIAN TUDOREL OLARU3*, LAURA ILEANA SOCEA1, LUCIAN ISCRULESCU1, GABRIEL SARAMET4, FLORICA BARBUCEANU5, ELENA MIHAELA PAHONTU6, STELIAN BARAITAREANU7, STEFANIA FELICIA BARBUCEANU1 1 Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, Organic Chemistry Department, 6 Traian Vuia Str., 020956, Bucharest, Romania 2 Military Medical Scientific Research Center, 24-28 Gr. Cobalcescu Str., 010195, Bucharest, Romania 3 Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, Pharmaceutical Botany and Cell Biology Department, 6 Traian Vuia Str., 020956, Bucharest, Romania 4 Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, Pharmaceutical Technology and Biopharmacy Department, 6 Traian Vuia Str., 020956, Bucharest, Romania 5 Institute for Diagnosis and Animal Health, 63 Dr. Staicovici Str., 050557, Bucharest, Romania 6 Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, General and Inorganic Chemistry Department, 6 Traian Vuia Str., 020956, Bucharest, Romania 7 University of Agronomical Sciences and Veterinary Medicine of Bucharest, Faculty of Veterinary Medicine, Clinical Sciences Department, 105 Splaiul Independentei, 050097, Bucharest, Romania

The heterocyclic compounds with oxazole nucleus arouse the interest of many researchers both in terms of their synthesis and physico-chemical properties, as well as of their biological potential, among them, the oxazolones occupying a special place. Oxazol-5(4H)-ones, also known as azlactones, are considered saturated or unsaturated, depending on the nature of the exocylic bond from the 4 th atom of the nucleus. Of these, unsaturated oxazolones present a particular importance both for the organic synthesis, as intermediates in the obtaining of other classes of compounds, as well as for their biological properties [1,2]. A large number of derivatives from this class are reported in the literature as having biological activity, such as antitumoral [3,4], antibacterial [5][6][7][8][9][10], antifungal [5,8,9], anti-inflammatory [11], antioxidant [5,12], tyrosinase inhibitors [13,14].
Motivated by these literature data, we initiated the synthesis of a new class of heterocyclic compounds from unsaturated oxazolones and of some triazinones derived from these, which to contain a diarylsulfone fragment in the same molecule, in order to obtain new derivatives with biological potential.
In a first stage, we investigated the cytotoxicity of the new synthesized compounds using Daphnia magna and Artemia salina organisms. Among other screening methods alternative to vertebrate animals, these bioassays are rapid, simple, reproducible and cost-efficient. Various studies demonstrated the usefulness of this method in testing various biological end-points [32][33][34][35][36].

Experimental part Chemistry
The reactants and solvents used in the synthesis were purchased commercially with high purity and were used without further purification. The melting points of the compounds were determined with a Böetius apparatus and are uncorrected. The elemental analysis was determined using a ECS-40-10 Costech micro-dosimeter. The FT-IR spectra were registered on a Bruker Vertex 70 spectrometer (in KBr pellets). The intensity of IR absorption bands are given as: very strong (vs), strong (s), medium (m) and weak (w). The NMR spectra were recorded on a Varian Gemini 300BB spectrometer at 300 MHz for 1 H-NMR and 75 MHz for 13 C-NMR. The DMSO-d 6 was used as solvent. The chemical shifts ä are reported in ppm relative to tetramethylsilane (TMS) internal standard and the coupling constants J are expressed in hertz Hz. The abbreviations for multiplicities and descriptors of signals used in the 1 H-NMR spectra are the following: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet, br = broad signal. For the acquisition of the mass spectra of compounds a APCI (Atmospheric Pressure Chemical Ionization) mass spectrometer was used. For oxazolones, a solution of about 0.5 mg /mL in chloroform was prepared and then was diluted 10 times with methanol (1% formic acid) for positive ionization, when protonated molecular ions were obtained [M+H] + . For triazinones a 0.5 mg/mL solution was diluted 10 times with methanol (1% ammonium carbonate) for negative ionization [M-H] -. The solutions of the oxazolones series were passed through the APCI interface with positive ionization and the triazinones solutions were passed through the APCI interface with negative ionization. A solution injection system with a loop mounted on a Reodyne 7725 valve and a Varian Prostar 240 SDM pump operating at a flow rate of 50 µL/min was used to drive the solutions. Protonated molecular ions (in positive ionization) or negative (by removing an acidic proton) were fragmented into the argon collision cell at 1.5mTorr. The fragments were scanned with the third quadrupole.
The chemical structures of the new compounds were confirmed by FT-IR, 1 H-NMR, 13 C-NMR, MS spectra and elemental analysis.
A mixture of N-acylated glycine 4a-c (5 mmol), 4iodobenzaldehyde (5 mmol) and sodium acetate (0.41 g) in acetic anhydride (1.8 mL) was refluxed for 4 h with magnetic stirring. To the obtained solution, 2 mL of ethanol was added and allowed it to stand overnight at cold. The obtained precipitate was filtered off, washed with boiling water and cold ethanol, when a yellow coloured crystalline compound was obtained. The compound was recrystallized from an ethanol-chloroform mixture (1:2, v:v).

Cytoxicity evaluation
The cytotoxicity bioassay was performed for compounds 5a-c, 6a,b and intermediates 4a-c.
Both experiments were carried out in controlled environment at 25±1°C, using a 16-h photoperiod and 8 h of darkness using a climatic chamber (Sanyo MLR-351H; Sanyo, San Diego, CA, USA).

Daphnia magna bioassay
The bioassay was performed in duplicate according to the method described in our previous research [41]. Each determination was carried out in PP tissue culture wells (Greiner Bio-One) using 10 organisms in each well at a final volume of 4 mL/sample and a final concentration of DMSO of 1%, used as negative control. Seven compounds were tested at 6 concentration levels ranging from 8 to 198 ìg/mL. The lethality (L%) was recorded over a 48h period of exposure at two points: 24 and 48h. The 50% lethal concentrations (LC50) were computed for each compound by interpolation on lethality curves plotted between L% values and the logarithm of concentrations using the least square fit method. The LC50 and 95% confidence intervals (CI95%) of LC50 were also calculated using the same method. All calculations were performed using the software GraphPad Prism v 5.1. The prediction of LC50 48h was performed using the GUSAR software application.

Artemia salina bioassay
Brine shrimp lethality test was conducted according to the protocol described by Meyer et al.
[36] with some modifications [42]. A. salina cysts were incubated in artificial sea water (40 g/L salinity) for 24h under continuous aeration. The newly hatched nauplii were separated from the shells, transferred to fresh sea water and incubated for 24 h. Each determination was carried out in PP tissue culture with 24-wells (Greiner Bio-One) using 10 organisms in each well at a final volume of 1 mL/sample and a final concentration of DMSO of 1%, used as negative control. The compounds were tested at the same levels of concentration as D. magna. The lethality (L%) was recorded over a 24h period of exposure and the calculations were made as presented above. No predictions could be performed for brine shrimps.

Results and discussions Chemistry
The key precursor, 2-(4-(4-X-phenylsulfonyl) benzamido) acetic acids 4, previously reported in the literature [37,38], were obtained following the Steiger procedure consisting of N-acylation of glycine with 4-(4-X-phenylsulfonyl)benzoyl chlorides 3. The simultaneous condensation and cyclization reaction of N-acylated glycine derivatives 4 with 4-iodobenzaldehyde, in acetic anhydride and in the presence of sodium acetate, led to the formation of new compounds from 4-iodobenzylidene-oxazole-5(4H)-ones 5. The 1,2,4-triazin-6(5H)-ones 6 resulted through a ringopening and recyclization reaction of the oxazolones 5 with phenylhydrazine, in acetic acid and sodium acetate. The heterocyclic compounds were obtained in moderate yields, in a relatively short reaction time. The spectral data validated the structures of these compounds.

IR Spectral data
The reaction between N-acylated glycine 4 and 4iodobenzaldehyde is firstly confirmed by the disappearance of the NH absorption band from the IR spectra of new compounds 5 that was present at 3416 cm -1 (4a), 3351 cm -1 (4b) and 3341 cm -1 (4c) in precursors 4 [37,38]. The C=O lactone group from oxazolones 5 is highlighted in the IR spectra of these compounds by absorption bands due to stretching vibration, νC=O, at 1793-1796 cm -1 and 1769-1772 cm -1 according to the literature for similar derivatives [39,40]..
The obtaining of triazinones from reaction of oxazolones with phenylhydrazine is firstly confirmed by appearance in the IR spectra of compounds 6 of a new absorption band at 3281 cm -1 in case of derivatives 6a,b and at 3282 cm -1 in 6c, characteristic to stretching vibration of NH group. The absorption band due to the stretching vibration of the C=O group appears at 1722 cm -1 for triazinones 6a,c and at 1723 cm -1 for triazinone 6b.

NMR Spectra data
The 1 H-NMR spectra of compounds 5 showed a singlet signal characteristic of =CH proton, with δ in the range In the 1 H-NMR spectra of the compounds 6, the presence of a new signal of NH proton from phenylhydrazine moiety, at ~ 9.00 ppm, is the most important evidence that the reaction of oxazolone with phenylhydrazine occurred.
In the 13 C-NMR spectra of the compounds 5, the carbon atoms from oxazole ring were highlighted by the following signals that appeared at the corresponding chemical shifts: 161.86-161.62 ppm for C-2, 132.92-133.47 ppm for the >C=CH (C-4) and 166.25-166.46 ppm for C=O (C-5), the last signal being the most deshielded, as expected.
In the 13 C-NMR spectra of triazinones 6, the C-3 atom resonated at ä=159.96-159.99 ppm, while the >C=CH (C-5) at 136.58-136.63 ppm. The C=O signal on the triazinone ring is observed in the range 166.70-168.77 ppm. Also, the methyne carbon from oxazolones 5 resonated at 131.22-132.62 ppm while the same carbon from the same position of triazinones 6 was appeared in region 128.54-128.67 ppm.

Mass spectra
Another support for the assigned structure of new compounds from oxazolones and triazinones classes was obtained by recording the mass spectra.
The mass spectra showed the presence of the molecular ions, thus confirming the structures of theese compounds. Fragmentation of oxazolones generally occurs by obtaining a carbonyl ion, stabilized by conjugation. For triazinones, the most characteristic fragmentation is the elimination of a neutral fragment with the formula C 6 H 5 N.
For Cl and Br atoms, isotopic contributions were taken into account.

Cytotoxicity evaluation Daphnia magna bioassay
The results are presented in table 1 and figure 3. At 24 h, the lethality induced by the tested compounds was below 30% and LC50 couldn't be calculated. After 48h of exposure, 4b and 4c did not produced aby lethal effects and no further calculations were possible. After calculating the LC50, the highest toxicity was exhibited by 5a and the lowest by 5b. Among the tested compounds, 5a had a LC50 lower than 100µg/mL, thus being a potent compound with biological activity on D. magna.
No correlation was found between GUSAR prediction values and experimental LC50 values. Moreover, 5b, 4b and 4c were found not to be toxic or exhibiting low toxicity, whereas the prediction software indicates them as potent cytotoxic compounds. By applying the t-student test on the two sets of data, predicted and experimentally obtained values, no similarity was found, p value being < 0.0001 which indicate a significant difference. A slightly correlation was found for compounds 4a, and 4b regarding the order of magnitude.

Artemia salina bioassay
The toxicity at 24h on A. salina was lower than 25% for all compounds at the tested concentrations. The results are comparable to those obtained at D. magna bioasay. As showed previously [42], the brine shrimp lethality test is less sensitive than D. magna, mainly due to a better resistance of A. salina. Further tests will be performed to evaluate the toxicity of the compounds at 48 h.
The cytotoxicity bioassays on Daphnia magna and Artemia salina indicated that among the tested compounds oxazolone 5a showed moderate to high cytotoxicity on D. magna, thus being a good candidate for further biological activity screening, including anticancer and antimicrobial.