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Synthesis and Characterization of New Series of 1,3-5-Triazine Hydrazone Derivatives with Promising Antiproliferative Activity

A new series of s-triazine hydrazone derivatives was prepared based on the reaction of 6-hydrazino-2,4-disubstituted-s-triazine with p-substituted benzaldehyde derivatives using a straightforward synthetic pathway. The antiproliferative activity of all synthesized compounds was evaluated against two human cancer cell lines; breast cancer MCF-7 and colon carcinoma HCT-116 using MTT assay. Among all, 11 compounds have shown strong to moderate antiproliferative activity with IC50 values in the range 1.01–18.20 µM in MCF-7 and 0.97–19.51 µM in HCT-116. The best results were obtained with 4,4’-(6-(2-(pyridin-2-ylmethylene)hydrazinyl)-1,3,5-triazine-2,4-diyl) dimorpholine 11 (IC50 = 1.0 µM and 0.98 µM in MCF-7 and HCT-116 cell lines, respectively). The substituents on the s-triazine core as well as the substituent at the benzylidene moiety have a great effect on the antiproliferative activity. Whereas compounds containing dimorpholino-s-triazine derivatives 8a–e showed more potent antiproliferative in MCF-7 compared to their analogs 7a–f (compounds containing two-piperidine rings), compounds containing one piperidine and one morpholine ring 9a–f showed better IC50 values in the range 10.4–22.2 µM. On the other hand, compounds containing two-piperidine rings 7a–f showed more potent antiproliferative in HCT-116 (IC50 values in the range 8.8–19.5 µM) than their analogs 8a–e and 9a–f.


Introduction
s-Triazine (1,3,5-triazine) is a heterocyclic compound, which is quite stable and its derivatives have wide practical applications in numerous fields. The most important reagent for obtaining these derivatives is cyanuric chloride (2,4-6-trichloro-1,3,5-triazine). This compound has provoked significant consideration due to its low cost, commercial availability, and considered as an ideal starting material in stepwise nucleophilic substitution for the synthesis of symmetric and nonsymmetric s-triazine derivatives at different temperatures [1][2][3].
The s-triazine scaffold affords the basis for the design and synthesis of various biologically active compounds with widespread applications in medicinal chemistry [4][5][6][7][8][9][10][11]. On the other hand, many researchers made notable progress on the design, synthesis, and evaluation of numerous s-triazine derivatives with great and promising results for the further development of new antitumor agents [12][13][14][15][16][17][18][19][20]. Recently, a series of s-triazine hydrazino derivatives I (Figure 1) was reported as selective inhibitors of the mammalian target of rapamycin (mTOR) [21]. The results showed that the phenolic hydroxyl group has a critical effect, while its analogs methoxy derivative showed a dramatic loss in the activity [21]. Later, another series of s-triazine hydrazone derivatives was reported with their particular target inhibitors to both epidermal growth factor (EGFR) and mutant epidermal growth factor tyrosine kinase receptors (EGFR TKs) [22]. Of these derivatives, the fluoro derivative II (Figure 1) showed the most potent activity against epidermal growth factor receptor (EGFR). Moreover, it exhibited considerable antiproliferative activity against A549, A431, and NCIH1975 cell lines. exhibited considerable antiproliferative activity against A549, A431, and NCIH1975 cell lines.
In a recent study [23], we reported the synthesis and evaluation of the antiproliferative activities of several s-triazine hydrazone derivatives III (Figure 1). The results showed that the substituents on the s-triazine ring especially, methoxy group and piperidine ring conferred greater selectivity for human liver cancer cell lines (HepG2), while the presence of the morpholine and piperidine ring exhibited greater selectivity for adenocarcinomic human alveolar basal epithelial cells (A549) with a reasonable inhibitory effect on HepG2 cells [23].
Later, we reported another series of s-triazine hydrazone derivatives that encompasses s-triazine and (2 or 4)-hydroxylbenzylidene derivatives IV and V (Figure 1) with their anticancer activity [24]. The results revealed that the position of the hydroxyl group on the benzylidine ring as well as the substituent on the s-triazine moiety have a great effect on the anticancer activity against breast cancer cells (MCF-7) and colon cancer (HCT-116).
Based on the reported results by our group and others, s-triazine, morpholine, and piperidine specifically serve as templates of a number of clinically used drugs [25], we report here the synthesis of a small library of compounds bearing the s-triazine, morpholine and piperidine core with benzylidene derivatives through the hydrazone linkage. The antiproliferative activity of this new series against breast cancer MCF-7 and colon carcinoma HCT-116 cell lines were evaluated as well.

Chemistry
The target products 7a-f, 8a-e, 9a-f, and 10-12 were synthesized in three steps as follows. As a first step, cyanuric chloride was reacted with different amines according to the reported method [26] to afford 2-chloro-4,6-disubstituted-s-triazine derivatives in good yields and purities. The spectral In a recent study [23], we reported the synthesis and evaluation of the antiproliferative activities of several s-triazine hydrazone derivatives III (Figure 1). The results showed that the substituents on the s-triazine ring especially, methoxy group and piperidine ring conferred greater selectivity for human liver cancer cell lines (HepG2), while the presence of the morpholine and piperidine ring exhibited greater selectivity for adenocarcinomic human alveolar basal epithelial cells (A549) with a reasonable inhibitory effect on HepG2 cells [23].
Later, we reported another series of s-triazine hydrazone derivatives that encompasses s-triazine and (2 or 4)-hydroxylbenzylidene derivatives IV and V ( Figure 1) with their anticancer activity [24]. The results revealed that the position of the hydroxyl group on the benzylidine ring as well as the substituent on the s-triazine moiety have a great effect on the anticancer activity against breast cancer cells (MCF-7) and colon cancer (HCT-116).
Based on the reported results by our group and others, s-triazine, morpholine, and piperidine specifically serve as templates of a number of clinically used drugs [25], we report here the synthesis of a small library of compounds bearing the s-triazine, morpholine and piperidine core with benzylidene derivatives through the hydrazone linkage. The antiproliferative activity of this new series against breast cancer MCF-7 and colon carcinoma HCT-116 cell lines were evaluated as well.

Chemistry
The target products 7a-f, 8a-e, 9a-f, and 10-12 were synthesized in three steps as follows. As a first step, cyanuric chloride was reacted with different amines according to the reported method [26] to afford 2-chloro-4,6-disubstituted-s-triazine derivatives in good yields and purities. The spectral data were in good agreement with the reported ones in literature [26]. As a second step, 2-chloro-4,6-disubstituted-s-triazine derivatives reacted with hydrazine hydrate in ethanol following the reported method [26,27] to afford the hydrazino derivatives 2-4 as a white solid in good yields and purities which have been used directly into the next step. Finally, 6-hydrazino-2,4-disubstituted-s-triazine derivatives 2-4 were reacted with p-substituted benzaldehyde derivatives 5a-f or 2-pyridinecarboxaldehyde 6 in the presence of the catalytic amount of acetic acid (AcOH) and ethanol as solvent to give the target products 7a-f, 8a-e, 9a-f, and 10-12 (Scheme 1, Table 1). The structure of the target products was confirmed by elemental analysis, FTIR, 1 H-NMR, and 13 C-NMR spectra, (Supplementary Materials, Figures S1-S16).
Molecules 2020, 25, x FOR PEER REVIEW 3 of 11 data were in good agreement with the reported ones in literature [26]. As a second step, 2-chloro-4,6disubstituted-s-triazine derivatives reacted with hydrazine hydrate in ethanol following the reported method [26,27] to afford the hydrazino derivatives 2-4 as a white solid in good yields and purities which have been used directly into the next step. Finally, 6-hydrazino-2,4-disubstituted-s-triazine derivatives 2-4 were reacted with p-substituted benzaldehyde derivatives 5a-f or 2pyridinecarboxaldehyde 6 in the presence of the catalytic amount of acetic acid (AcOH) and ethanol as solvent to give the target products 7a-f, 8a-e, 9a-f, and 10-12 (Scheme 1, Table 1). The structure of the target products was confirmed by elemental analysis, FTIR, 1 H-NMR, and 13 C-NMR spectra.

Scheme 1.
Synthetic route for preparation of the target products.

Antiproliferative Activity
The antiproliferative activity of the synthesized s-triazine derivatives 7a-f, 8a-e, 9a-f, and 10-12 were studied in human breast cancer (MCF-7) and colon carcinoma (HCT-116). Most of the s-triazine hydrazone derivatives affected the cell viability of the two cancer cell lines, as determined by the MTT cell viability assay. The results revealed that the most effective compound was 11 (compound containing two morpholine rings on the s-triazine core and pyridine at the hydrazone terminal) which affected the cell viability of the two cancer cell lines with IC 50 values 1.0 µM and 0.98 µM in MCF-7 and HCT-116, respectively, Figure 2). Its analogs compound 10 (compound containing two-piperidine rings) and 12 (compound containing piperidine and morpholine ring) showed less potent activities as shown in Table 2. Therefore, the substituent on the s-triazine ring has a great effect on the activity of the tested compounds. Compounds containing two morpholine rings 8a-e showed more potent antiproliferative activity in MCF-7 with IC 50 values in the range 13.4-29.3 µM compared to their analogous containing two piperidine rings 7a-f (IC 50 values in the range 11.5-39.9 µM).
in the range 10.4-22.2 µM. These data are in good agreement with our previously reported related striazine derivatives [24,27]. Interestingly, compounds containing two-piperidine rings 7a-f affected the cell viability of the HCT-116 (IC50 values in the range 8.8-19.5 µM) more than their analogs 8a-e (IC50 values in the range 18.3 > 50 µM) and 9a-f (IC50 values in the range 22-42.2 µM) as shown in Table 2.
On the other hand, the substituent on the benzylidene moiety has also a great impact on the antiproliferative activity. As shown in Table 2, p-fluoro benzylidene derivatives 7e, 8e, and 9e derivatives affected the cell viability of both cell lines in lower micromolar range compared to their analogs containing p-chloro (7b, 8b, and 9b) or p-bromo (7c, 8c, and 9c) benzylidene derivatives (Table 2). Thus, the presence of fluorine atoms significantly improves the pharmacological and physicochemical properties as the metabolic stability, lipophilicity as well as ligand binding [28,29], these results also agreed with the reported results by Bai et al. [22]. The replacement of fluorine atoms in compounds 7e, 8e, and 9e with by hydroxy group as in compounds 7d, 8d, and 9d showed also good antiproliferative activities against MCF-7 cell line with IC50 = 18.2, 14, and 10.4 µM, respectively ( Table 2). These results agreed with the reported results which indicated that the phenolic hydroxyl group acts as a hydrogen donor which improves the anticancer activity [21,24].   Compounds containing one morpholine and one piperidine ring 9a-f showed better IC 50 values in the range 10.4-22.2 µM. These data are in good agreement with our previously reported related s-triazine derivatives [24,27] Table 2.
On the other hand, the substituent on the benzylidene moiety has also a great impact on the antiproliferative activity. As shown in Table 2, p-fluoro benzylidene derivatives 7e, 8e, and 9e derivatives affected the cell viability of both cell lines in lower micromolar range compared to their analogs containing p-chloro (7b, 8b, and 9b) or p-bromo (7c, 8c, and 9c) benzylidene derivatives (Table 2). Thus, the presence of fluorine atoms significantly improves the pharmacological and physicochemical properties as the metabolic stability, lipophilicity as well as ligand binding [28,29], these results also agreed with the reported results by Bai et al. [22]. The replacement of fluorine atoms in compounds 7e, 8e, and 9e with by hydroxy group as in compounds 7d, 8d, and 9d showed also good antiproliferative activities against MCF-7 cell line with IC 50 = 18.2, 14, and 10.4 µM, respectively (Table 2). These results agreed with the reported results which indicated that the phenolic hydroxyl group acts as a hydrogen donor which improves the anticancer activity [21,24].

Materials and Methods
All solvents and reagents purchased from commercial suppliers and used without further purification. 1

General Method for the Synthesis of 1,3,5-Triazine-Hydrazone Derivatives
2-Hydrazino-4,6-disubstituted-1,3,5-triazine derivatives 2-4 were synthesized first following the synthetic strategy and methods reported by our group and others [20][21][22][23][24]. The target products were synthesized as follows: Aldehyde derivatives 5a-f or 6 (10 mmol) in ethanol (10 mL) were added to a solution of 2-4 in ethanol (20 mL) containing 2-3 drops of acetic acid at room temperature. After complete addition the reaction mixture refluxed for 4-6 h, and the progress of the reaction was followed by thin-layer chromatography (TLC) using ethyl acetate-hexane 2:1. The reaction left to cool down to room temperature and the solid product separated by filtration, washed with cooled ethanol, and then dried at room temperature.
The characterization of compounds 8a-d studied herein previously reported by our group [30]. The characterization of the remaining compounds described below.

In Vitro Antiproliferative Assay
The target products 7a-f, 8a-e, 9a-f, 10-12 were evaluated for antiproliferative activity using the MTT (Tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay in human breast cancer (MCF-7) and colon carcinoma (HCT-116) cell lines. Cells were seeded in triplicate in 96-well plates at a density of 10 × 10 3 cells/mL in a total volume of 200 µL per well. 0.1% of DMSO used as vehicle control. Each well was treated with 2 µL tested compounds, which had been pre-prepared as stock solutions in ethanol to furnish the concentration range of study, 10 nM to 100 µM, and re-incubated for a further 72 h. The culture medium was then removed and the cells washed with 100 µL phosphate-buffered saline (PBS) and 50 µL MTT added, to reach a final concentration of 1 mg/mL MTT added. Cells incubated for 2 h in darkness at 37 • C. At this point, solubilization was begun through the addition of 200 mL DMSO and the cells maintained at room temperature in darkness for 20 min to ensure thorough color diffusion before reading the absorbance. Plates were incubated for 72 h at 37 • C + 5% CO 2 . The MTT (5 mg/mL in PBS) was added and incubated for another 4 h, the optical density was detected with a microplate reader at 570 nm. Results expressed as percentage viability relative to vehicle control (100%). Dose-response curves plotted and IC 50 values (concentration of drug resulting in 50% reduction in cell survival) were obtained using the commercial software package Prism (GraphPad Software, Inc., La Jolla, CA, USA). All the experiments were repeated in at least three independent experiments.

Conclusions
The s-triazine hydrazone derivatives reported herein were evaluated for their inhibitory effect of the growth of two human cancer cell lines; breast cancer MCF-7 and colon cancer HCT-116 using MTT assay. Compound 11 which containing two morpholine rings on the s-triazine core and pyridine at the hydrazone terminal showed the greatest antiproliferative activity amongst all the tested compounds (IC 50 = 1.0 µM and 0.98 µM in MCF-7 and HCT-116, respectively). The substituent on the s-triazine ring has a great effect on the activity of the tested compounds. This was observed with compound 11 vs. 10 (IC 50 = 32.8 µM in MCF-7 and >50 µM in HCT-116) and 12 (IC 50 = 17.7 µM in MCF-7 and 30.4 µM in HCT-116). This also was noticed in the series of compounds containing two morpholine rings 8a-e which showed more potent antiproliferative activity in MCF-7 (IC 50 values in the range 13.4-29.3 µM) compared to their analogous containing two-piperidine rings 7a-f (IC 50 values in the range 11.5-39.9 µM). While compounds containing one morpholine and one piperidine rings 9a-f showed better IC 50 values in the range 10.4-22.2 µM, these results agreed with the reported in the literature [24,27]. Interestingly, compounds containing two-piperidine rings 7a-f showed more potent activity in HCT-116 cell line (IC 50  The substituent on the benzylidene moiety also affected the antiproliferative activity, where derivatives with p-fluoro derivatives (7e, 8e, and 8e) exhibited more potent activities in both tested cell lines (IC 50 = 11.5, 13.4, 13.9 µM in MCF-7 and 14, 18.3, 22 µM in HCT-116, respectively) than their analogs p-chloro (7b, 8b, and 9b) and p-bromo (7c, 8c, and 9c) derivatives. Replacement of the fluorine atom by hydroxy group showed better anticancer activities compared to its analogous methoxy group.
Taken together, these results efforts on the synthesis of more new series based on s-triazine hydrazone derivatives with different groups in progress in our lab, proving the antiproliferative activity and possible mechanism of action of s-triazine hydrazone derivatives which might be of special interest in medicinal chemistry.