Synthesis , X-Ray Crystal Structures , and Preliminary AntiproliferativeActivitiesofNews-Triazine-hydroxybenzylidene Hydrazone Derivatives

Chemistry Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Alexandria 21321, Egypt Department of Chemistry, Faculty of Science, Mansura University, Mansura 35516, Egypt Department of Pharmacognosy, Faculty of Pharmacy, Mansura University, Mansura 35516, Egypt Department of Medicinal Chemistry, Faculty of Pharmacy, Mansura University, Mansoura 35516, Egypt

Similarly, s-triazine derivatives represent important class of compounds in medicinal chemistry [20][21][22][23].Cyanuric chloride has been used as building blocks in the synthesis of vast derivatives bearing the s-triazine moiety, due to the low-cost, commercial availability, and ease of displacement of the three chlorine atoms by various nucleophiles under controlled temperature [24].
e results showed that s-triazine with the two substituents methoxy and piperidine in the target product made the compound more selective to the hepatocyte carcinoma HepG2 (IC 50 value of 1.5 µg/mL).On the contrary, the combination between morpholine and piperdine motifs in the final target made the compound more selective to the lung carcinoma A549 (IC 50 value of 5.6 µg/ml) and with reasonable effect to the hepatocyte carcinoma HepG2 (IC50 value 6.5 µg/ml) [44], Recently, Bai et al. [45] identified a series of 1,3,5-triazine hydrazone derivatives as dualeffective inhibitors against both epidermal growth factor receptor (WTEGFR) and mutant epidermal growth factor receptor (EGFR).Moreover, some of them exhibited considerable antiproliferative activity against A549, A431, and NCIH1975 cell lines.
ey claimed that the phenolic hydroxyl group in the series is critical in the activity because it acts as a hydrogen donor, where when it was substituted by a methoxy, a dramatic loss in the activity was observed.
ese results encouraged us to develop small library (Figure 1) bearing of s-triazine derivatives with different substituents and (2 or 4)-hydroxybenzylidene derivatives through a hydrazone linkage.e antiproliferative activities of the target products will be discussed to fine-tune and get more information related to the effect of both substituent in the s-triazine moiety and the position of the hydroxyl group in the benzylidene core attached to the s-triazine through the hydrazone linkage.

Experimental Section
2.1.Chemistry 2.1.1.General.All reagents and solvents were purchased from commercial suppliers and were used without further purification; NMR spectra ( 1 H-NMR and 13 C-NMR) were recorded on a JEOL 400 MHz spectrometer. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) were run in either deuterated dimethylsulphoxide (DMSO-d 6 ) or deuterated chloroform (CDCl 3 ).Chemical shifts (δ) are referred in terms of ppm and J-coupling constants are given in Hz.Mass spectra were recorded on a JEOL of JMS-600 H. Elemental analysis was carried out on an Elmer 2400 elemental analyzer.All melting points were measured on a Gallenkamp melting point apparatus in open glass capillaries and are uncorrected.IR Spectra were measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer.
e compounds of 4f, 5b, and 5f obtained as single crystals by slow evaporation from ethanol solution to afford the pure compounds at room temperature.Data were collected on a Bruker APEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Mo Kα radiation, λ � 0.71073 Å at 293 (2) and 296 (2) K. Cell refinement and data reduction were carried out by Bruker SAINT.SHELXT was used to solve structures [52,53].e final refinement was carried out by full-matrix least-squares techniques with anisotropic thermal data for nonhydrogen atoms on F. CCDC No. 1567719 (4f ), 1567728 (5b), and 1567725 (5f ) containng the supplementary crystallographic data for these compounds wwhich can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.

General Procedure for the Synthesis of Schiff Bases 4a-i and 5a-h.
A solution of hydroxybenzaldehyde derivatives 3a-b (6 mmol), drop of acetic acid, and s-triazine derivatives 2a-i (6 mmol) in EtOH (10 ml) were mixed and heated under reflux for 3 h (TLC 20% EtOAc/n-hexane).e solvent was evaporated slowly, to provide the corresponding product 4ai and 5a-h.Cl Cl Scheme 1: Synthetic route for preparation of s-triazine Schi -base derivatives.

Anticancer Activity.
e cytotoxic activity of 18 newly synthesized compounds was tested against two mammalian cancer cell lines, colon cancer cells (HCT-116), and breast cancer cells (MCF-7).
e cells were seeded in 96-well plates as 5 × 10 4 cells/mL (100 µl/well).Six serial dilutions of tested compounds were added after overnight incubation of the cells at 37 °C and 5% CO 2 .DMSO was used as a negative control (0.5 %). e cells were incubated for 48 hrs.After that, 10 µl of MTT (5 mg/ml PBS) was added to each well and incubated for another 4 hours.
e formazan crystals were solubilized by 100 µl acidified SDS solution (10% SDS/0.01M HCl).Biotek ® plate reader measured the absorbance after 14 hours of incubation at 37 °C and 5% CO 2 at 570 nm.Each experiment was repeated three times, and standard deviation was calculated (±).IC 50 was calculated as the concentrations that cause 50% inhibition for cell growth.

Results and Discussion
3.1.Chemistry.e s-triazine Schiff-base derivatives 4a-i and 5a-h were synthesized in three steps: (i) one pot-reaction of cyanuric chloride with the first amine in acetone-water media and in the presence of Na 2 CO 3 at 0-5 °C for 2 h.e second amine was added to the reaction mixture followed by addition of equivalent amount of Na 2 CO 3 at 5 °C and then let the reaction mixture reach the room temperature under stirring for 18 h to afford 6-chloro-2,4-disubstituted-s-triazine derivatives in good yields and purities; the spectral data were in good agreement with the reported in the literature [44].(ii) 6chloro-2,4-disubstituted-s-triazine derivatives reacted with hydrazine hydrate in ethanol according to the reported method [47,49], to afford the hydrazino derivatives 2a-i as white solid in good yields and purities which have been used directly in the next step without further purification.(iii) 6hydrazino-2,4-disubstituted-s-triazine 2a-i were reacted with substituted aldehydes 3a,b in ethanol under reflux in the presence of catalytic amount of AcOH to give the target products 4a-i and 5a-i in high yields (Scheme 1).
e final products were separated by simple filtration after the precipitation.
e molecular structure were deduced by different set of physical spectrophotometric tools including H-NMR, C-NMR, LC-MS, IR, and CHN elemental analysis.Of these, the molecular structure of three compounds, namely, 4f, 5b, and 5f, has been further confirmed by X-ray single crystal diffraction technique (Figure 2).
In the title compounds, the crystallographic data and refinement information are summarized in Figures S1-S6 and Tables S1-S9 (Supplementary Materials).
e asymmetric unit of the three containing is shown in Figure 2, where compound 4f contains an additional lattice water molecule.
All the bond lengths and angles are in normal ranges [55].
In the crystal packing, the molecules of compound

Biological Activity.
Antiproliferative activity for the new synthesized s-triazine Schiff-base derivatives was tested against two cancer cell lines for breast cancer (MCF-7) and colon cancer (HCT-116).An initial screening for the possible antiproliferative activity was carried out at 10 µM for 48 hours, and then the viability was determined by MTT assay.Among 18 tested compounds, only compounds 4a and 5a showed cytotoxic activity against breast cancer cells at 10 µM (Figure 3).On the contrary, very weak activity was observed for the tested compounds against colon cancer cells (Figure 4).
Based on the results obtained and showed in Figures 3  and 4, we can concluded that 2-hydroxybenzylidene derivatives showed better activity than the analogous 4hydroxybenzylidene 5a-h derivatives vs. 4a-h derivatives even in the weak activity.is might be due to in the 4position the hydroxyl group is more sterically hindered, while when the hydroxyl group in the 3-position, it become less sterically hindered and more available for hydrogen donor, also has more inductive on the benzylidene ring, which usually preferred in most of the cases as previously reported [46,49].In the meantime, the substituents in the s-triazine ring have affected the activity of the prepared compounds.e substituent with two piperidine ring always showed higher reactivity than the same with two morpholine ring as shown in cases 4a and 5a vs. 4b and 5b.e same effect of piperidine was noticed in the cases of 5e vs. 5b and 4c vs. 4i (which they have weak activity).On the contrary, the presence of two morpholine rings showed better activity than derivatives with only one as in the cases 4b vs. 5c, 5d, 5f, 5g, and 5h.ese observations agreed with our previously reported data [44].
As noticed from Figure 5, the concentration that kills 50% of breast cancer cells was evaluated for the two most active compounds 4a and 5a.Compound 4a showed IC 50 of 27 µM (13.3 µg/mL), while 5a was more active at IC 50 of 17 µM (8.4 µg/mL) which was very close to the chemotherapeutic drug cisplatin which killed 50% of the cells at concentration of 20 µM (6 µg/mL).

Conclusions
e new series of s-triazine Schiff-base derivatives were synthesized using the mild and conventional method.e chemical structures of the prepared compounds have been confirmed by different sets of spectroscopic techniques.e newly compounds examined against antiproliferative activity showed that two derivatives have piperidine moiety more selective against the two cell lines (MCF-7) and (HCT-116).Based on the results obtained, we can conclude that both the substituent on the triazine ring and the position of the hydroxy group in the benzylidene moiety have critical e ect on the biological activity of the prepared compounds, where 2-hydroxybenzylidene derivatives showed better activity than the analogous 4-hydroxybenzylidene 5a-h derivatives vs. 4a-h derivatives even in the weak activity cases.e substituents in the s-triazine ring have a ected the activity   Journal of Chemistry where s-triazine with two piperidine ring always showed higher reactivity than analogous one two morpholine ring as shown in case 4a and 5a vs. 4b and 5b.e concentration that kills 50% of breast cancer cells was evaluated for the two most active compounds 4a and 5a.Compound 5a showed higher activity (IC 50 � 17 µM (8.4 µg/mL)) than 4a (IC 50 � 27 µM (13.3 µg/mL)).ese values, especially those of 5a, are very close to that of the chemotherapeutic drug cisplatin, having IC 50 of 20 µM (6 µg/mL).Finally, we propose that the antiproliferative activity of these two compounds (4a and 5a) deserves further attention for additional development as potent anticancer agents.