# Synthesis, X-Ray Crystal Structures, and Preliminary Antiproliferative Activities of New s-Triazine-hydroxybenzylidene Hydrazone Derivatives

Authors: Assem Barakat; Fardous F. El-Senduny; Zainab Almarhoon; Hessa H. Al-Rasheed; Farid A. Badria; Abdullah Mohammed Al-Majid; Hazem A. Ghabbour; Ayman El-Faham
Journal: Journal of Chemistry
 (2019)

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## Abstract

We herein report a new small library of Schiff-base compounds that encompasses s-triazine and (2 or 4)-hydroxylbenzylidene derivatives. These compounds were synthesized through a hydrazone linkage connecting both the s-triazine and hydroxybenzylidene derivatives. The synthetic strategy adopted allowed the synthesis of the target compounds with excellent yields and purities as observed from their NMR (1H and 13C) and elemental analysis. Furthermore, 4f, 5b, and 5f were further confirmed by X-ray single crystal diffraction technique. The preliminary antiproliferative activities for the synthesized compounds were tested against two different cancer cell lines including breast cancer (MCF-7) and colon cancer (HCT-116). From the eighteen compounds, which have been examined, only two derivatives having piperidine moiety showed more selectivity against the two cell lines MCF-7 and HCT-116, while the others showed very weak activity. The position of the hydroxyl group in the benzylidine ring and the substituent on the s-triazine moiety has great effect on the activity of the prepared compounds. The IC50 values for the two derivatives 4a and 5a evaluated against breast cancer cells, very close to those for the chemotherapeutic drug cisplatin, are 27 µM (13.3 µg/mL), 17 µM (8.4 µg/mL), and 20 µM (6 µg/mL) for 4a, 5a, and cisplatin, respectively. These results propose the preliminary antiproliferative activity of these two derivatives may deserve further consideration for development of new derivatives as potent anticancer agents.


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## Body

## 1. Introduction

Schiff bases have gained great importance in which these compounds have been found to exhibit antimicrobial [1–4], antiviral [1, 5], antioxidant [6], and antitumor [7, 8] activities. These activities are largely attributed due to the presence of the azomethine (R-NH-N=C-R) group which is important synthon for several transformations [9, 10]. In addition, the hydrazone linkage provides an appropriate system for pH-dependent of drugs release [11]. Several hydrazone derivatives have shown to exhibit antiproliferative activities with the ability to prevent cell progression in cancerous cells through different mechanisms [12]. Several hydrazone derivatives have been also shown antitumoral [13], antifungal, antiplatelet, antitubercular, antiviral, anticonvulsant, antimalarial, anti-inflammatory, and antipyretic activities [9, 14–19].Similarly, s-triazine derivatives represent important class of compounds in medicinal chemistry [20–23]. 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]. These advantages allowed for the preparation of mono-, di- and trisubstituted s-triazines derivatives with a wide range of biological activities [25–27], such as adenosine receptor antagonist [28], antiamoebic [29], antileishmanial [31–33], anticancer [32], antimalarial [34], antimicrobial [35–37], antiviral [38], antitubercular [39], carbonic anhydrase inhibitor [40], and cathepsin B inhibitor [41], Previously, we reported new series of s-triazine Schiff-base derivatives [42–44]; among of the reported compounds, only three derivatives were able to inhibit the growth of lung (A549) and hepatocellular carcinoma (HepG2) cancer cell lines. The 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 (IC50 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 (IC50 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 dual-effective 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.Menwear et al. [46] reported 1,3,5-triazine hydrazone based on 4-hydroxy-3,5-dimethoxyphenyl derivatives (Figure 1) as selective inhibitors of the mammalian target of rapamycin (mTOR). They 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.Figure 1
Rational design for the target products.These results encouraged us to develop small library (Figure1) bearing of s-triazine derivatives with different substituents and (2 or 4)-hydroxybenzylidene derivatives through a hydrazone linkage. The 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.


## 2. 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 (1H-NMR and 13C-NMR) were recorded on a JEOL 400 MHz spectrometer. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) were run in either deuterated dimethylsulphoxide (DMSO-d6) or deuterated chloroform (CDCl3). 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.


#### 2.1.2. 6-Chloro-4,6-disubstituted s-Triazine Derivatives (2a-i)

6-Chloro-4,6-disubstituted s-triazine derivatives (2a-i) were synthesized following the strategies and methods already reported by our group and others [47–51], as shown in Scheme 1. 6-Hydrazine-4,6-disubstituted s-triazine derivatives were synthesized following the published and reported method by our group [47, 49]. All spectral data were in good agreement with the reported data.Scheme 1
Synthetic route for preparation of s-triazine Schiff-base derivatives.The compounds of4f, 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]. The 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.


#### 2.1.3. General Procedure for the Synthesis of Schiff Bases4a-i and 5a-h

A solution of hydroxybenzaldehyde derivatives3a-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). The solvent was evaporated slowly, to provide the corresponding product 4a-i and 5a-h.(1) (E)-2,6-Di-tert-butyl-4-((2-(4,6-di(piperidin-1-yl)-1,3,5-triazin 2-yl)hydrazono)methyl)phenol (4a). Yellow powder in yield 93%; m.p: >250°C; IR (KBr, cm−1): 3637, 3423, 3197, 2960, 2935, 1583, 1570, 1510, 1462;1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.55 (m, 8H, 4CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 4H, 2CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 206.9, 156.6, 136.0, 128.9,124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 494.70 [M + 1]+; Anal. for C28H43N7O; Calcd: C, 68.12; H, 8.78; N, 19.86; Found: C, 68.13; H, 8.79; N, 19.89.(2) (E)-2,6-Di-tert-butyl-4-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4b). Yellow powder in yield 91%; m.p: 180°C; IR (KBr, cm−1): 3086, 3053, 2953, 2885,1598, 1560, 1516, 1463, 1371;1H-NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1H, CH =), 8.10 (brs, 1H, NH), 7.38 (s, 1H, Ph), 7.12 (s, 1H, Ph), 3.70 (brs, 8H, 4CH2), 1.50 (m, 8H, 4CH2), 1.35 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.9, 155.3, 135.1, 129.0, 125.1, 65.9, 44.6, 34.2, 30.9, 30.1LC/MS (ESI): 498.31 [M + 1]+; Anal. for C26H39N7O3; Calcd: C, 62.75; H, 7.90; N, 19.70; Found: C, 62.71; H, 7.91 N, 19.72.(3) (E)-2,6-Di-tert-butyl-4-((2-(4-(diethylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4c). Yellow powder in yield 89%; m.p: 195°C; IR (KBr, cm−1): 3420, 2999, 2960, 2908, 2870, 1624, 1591, 1456;1H-NMR (400 MHz, DMSO-d6): δ 8.60 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.66 (s, 1H, Ph), 7.50 (s, 1H, Ph), 3.82 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.49 (s, 9H, 3CH3), 1.44 (s, 9H, 3CH3), 1.18 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.4, 176.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.1, 12.3; LC/MS (ESI): 484.33 [M + 1]+; Anal. for C26H41N7O2; Calcd: C, 64.57; H, 8.54; N, 20.27; Found: C, 64.57; H, 8.53; N, 20.25.(4) (E)-2,6-Di-tert-butyl-4-((2-(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4d). Yellow powder in yield 80%; m.p: 114°C; IR (KBr, cm−1): 3215, 3045, 2958, 2908, 2866, 1614, 1598, 1544,1463;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 443.27 [M + 1]+; Anal. for C23H34N6O3; Calcd: C, 62.42; H, 7.74; N, 18.99; Found: C, 62.43; H, 7.75; N, 19.01.(5) (E)-2,6-Di-tert-butyl-4-((2-(4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4e). Yellow powder in yield 90%, m.p: >250°C; IR (KBr, cm−1): 3215,3157, 3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463;1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.50 (m, 4H, 2CH2), 1.45 (m, 4H, 2CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 496.33 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 65.43; H, 8.34; N, 19.78; Found: C, 65.43; H, 8.35; N, 19.76.(6) (E)-4-((2-(4-(Benzylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-2,6-di-tert-butylphenol (4f). Yellow powder in yield 88%, m.p: 169°C; IR (KBr, cm−1): 3421, 3116, 2958, 2860, 1618, 1591, 1508, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.37-7.28 (m, 5H, Ph), 7.19 (s, 1H, Ph), 4.23 (s, 2H, CH2Ph), 3.73 (brs, 4H, 2CH2), 1.50 (m, 4H, 2CH2), 1.40 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 65.9, 44.7, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 518.32 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 67.28; H, 7.59; N, 18.94; Found: C, 67.30; H, 7.60; N, 18.95.(7) (E)-2,6-Di-tert-butyl-4-((2-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4g). Yellow powder in yield 87%; m.p: 151°C; IR (KBr, cm−1): 3448, 3215, 3169, 2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.35 (s, 1H, CH =), 8.02 (brs, 1H, NH), 7.50 (s, 1H, Ph), 7.30-7.26 (m, 5H, Ph), 7.20 (s, 1H, Ph), 3.70 (brs, 4H, 2CH2), 1.56 (m, 4H, 2CH2), 1.35 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 66.0, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 504.30 [M + 1]+; Anal. for C28H37N7O2; Calcd: C, 66.77; H, 7.41; N, 19.47; Found: C, 66.76; H, 7.40; N, 19.49.(8) (E)-2,6-Di-tert-butyl-4-((2-(4-methoxy-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4h). Yellow powder in yield 93%; m.p: 124°C; IR (KBr, cm−1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3), 1.35 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 441.29 [M + 1]+; Anal. for C24H36N6O2; Calcd: C, 65.43; H, 8.24; N, 19.07; Found: C, 65.44; H, 8.23; N, 19.10.(9) (E)-2,6-Di-tert-butyl-4-((2-(4-(diethylamino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4i). Yellow powder in yield 94%; m.p: 169°C; IR (KBr, cm−1): 3423, 3197, 2960, 2908, 2858, 1583, 1570, 1510, 1435;1H-NMR (400 MHz, DMSO-d6): δ 8.44 (s, 1H, CH =), 7.95 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.47 (s, 1H, Ph), 3.76 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.49 (s, 9H, 3CH3), 1.44 (s, 9H, 3CH3), 128 (m, 2H, CH2), 1.22 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 199.3, 176.1, 156.2, 142.8, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.0, 12.2; LC/MS (ESI): 482.35 [M + 1]+; Anal. for C27H43N7O; Calcd: C, 67.32; H, 9.00; N, 20.36; Found: C, 67.31; H, 9.00; N, 20.35.(10) (E)-2,4-Di-tert-butyl-6-((2-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5a). Yellow powder in yield 90%; m.p: 217°C; IR (KBr, cm−1): 3637, 3423, 3197, 2960, 2935, 1583, 1570, 1510, 1462;1H-NMR (400 MHz, DMSO-d6): δ 8.03 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.32 (s, 1H, Ph), 7.01 (s, 1H, Ph), 3.79 (brs, 8H, 4CH2), 1.51 (m, 8H, 4CH2), 1.38 (s, 18H, 6CH3), 1.27 (m, 4H, 2CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 206.9, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 494.35 [M + 1]+; Anal. for C28H43N7O; Calcd: C, 68.12; H, 8.78; N, 19.86; Found: C, 68.11; H, 8.78; N, 19.83.(11) (E)-2,4-Di-tert-butyl-6-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5b). Yellow powder in yield 95%; m.p: 188°C; IR (KBr, cm−1): 3086, 3053, 2953, 2885,1598, 1560, 1516, 1463, 1371;1H-NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1H, CH =), 8.10 (brs, 1H, NH), 7.38 (s, 1H, Ph), 7.12 (s, 1H, Ph), 3.70 (brs, 8H, 4CH2), 1.50 (m, 8H, 4CH2), 1.35 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.9, 155.3, 135.1, 129.0, 125.1, 65.9, 44.6, 34.2, 30.9, 30.1; LC/MS (ESI): 498.31 [M + 1]+; Anal. for C26H39N7O3; Calcd: C, 62.75; H, 7.90; N, 19.70; Found: C, 62.75; H, 7.89; N, 19.72.(12) (E)-2,4-Di-tert-butyl-6-((2-(4-(diethylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5c). Yellow powder in yield 87%; m.p: 203°C; IR (KBr, cm−1): 3421, 3116, 2966, 2980, 1647, 1618, 1591, 1508,1499, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.82 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.53 (s, 1H, Ph), 7.54 (s, 1H, Ph), 3.82 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.57 (s, 9H, 3CH3), 1.40 (s, 9H, 3CH3), 1.18 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.4, 176.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.1, 12.3; 13C-NMR (100 MHz, DMSO-d6): δ = ; LC/MS (ESI): 484.33 [M + 1]+; Anal. for C26H41N7O2; Calcd: C, 64.57; H, 8.54; N, 20.27; Found: C, 64.56; H, 8.53; N, 20.24.(13) (E)-2,4-Di-tert-butyl-6-((2-(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5d). Yellow powder in yield 83%; m.p: 195°C; IR (KBr, cm−1): 3215, 3045, 2958, 2908, 2866, 1614, 1598, 1544,1463;1H-NMR (400 MHz, DMSO-d6): δ 7.95 (s, 1H, CH =), 7.78 (brs, 1H, NH), 7.65 (s, 1H, Ph), 7.50 (s, 1H, Ph), 3.92 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 443.27 [M + 1]+; Anal. for C23H34N6O3; Calcd: C, 62.42; H, 7.74; N, 18.99; Found: C, 62.42; H, 7.75; N, 19.22.(14) (E)-2,4-Di-tert-butyl-6-((2-(4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5e). Yellow powder in yield 86%; m.p: 171°C; IR (KBr, cm−1) 3134, 3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463; 1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.50 (m, 4H, 2CH2), 1.45 (m, 4H, 2CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 496.33 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 65.43; H, 8.34; N, 19.78; Found: C, 65.44; H, 8.35; N, 19.97.(15) (E)-2-((2-(4-(Benzylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-4,6-di-tert-butylphenol (5f). Yellow powder in yield 85%; m.p: 152°C; IR (KBr, cm−1): 3421, 3116, 2958, 2860, 1618, 1591, 1508, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.37-7.28 (m, 5H, Ph), 7.19 (s, 1H, Ph), 4.23 (s, 2H, CH2Ph), 3.73 (brs, 4H, 2CH2), 1.50 (m, 4H, 2CH2), 1.40 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 65.9, 44.7, 31.2, 30.0, 25.6, 24.5; 13C-NMR (100 MHz, DMSO-d6): δ = ; LC/MS (ESI): 518.32 [M + 1]+; Anal. For C27H41N7O2; Calcd: C, 67.28; H, 7.59; N, 18.94; Found: C, 67.29; H, 7.60; N, 19.06.(16) (E)-2,4-di-tert-butyl-6-((2-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5g). Yellow powder in yield 88%; m.p: 242°C; IR (KBr, cm−1): 3448, 3215, 3169, 2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.35 (s, 1H, CH =), 8.02 (brs, 1H, NH), 7.50 (s, 1H, Ph), 7.30-7.26 (m, 5H, Ph), 7.20 (s, 1H, Ph), 3.70 (brs, 4H, 2CH2), 1.56 (m, 4H, 2CH2), 1.35 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 66.0, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 504.30 [M + 1]+; Anal. for C28H37N7O2; Calcd: C, 66.77; H, 7.41; N, 19.47; Found: C, 66.77; H, 7.41; N, 19.48.(17) (E)-2,4-Di-tert-butyl-4-((2-(4-methoxy-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5h). Yellow powder in yield 81%; m.p: 155°C; IR (KBr, cm−1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3), 1.35 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 441.29 [M + 1]+; Anal. for C24H36N6O2; Calcd: C, 65.43; H, 8.24; N, 19.07; Found: C, 65.42; H, 8.25; N, 19.20.


### 2.2. Anticancer Activity

The 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). The cell lines were obtained from VACSERA, holding company for biological products and vaccines, Cairo, Egypt. The cells were grown at 37°C and 10% CO2 in DMEM (Lonza, 12-604F) medium supplemented with 10% fetal bovine serum (FBS, Lonza, Cat. No.14-801E), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Lonza, 17-602E). The viability of the cells were quantified by using MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which measures the activity of mitochondrial dehydrogenase in the viable cells [54].The cells were seeded in 96-well plates as 5 × 104 cells/mL (100 µl/well). Six serial dilutions of tested compounds were added after overnight incubation of the cells at 37°C and 5% CO2. DMSO was used as a negative control (0.5 %). The 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. The formazan crystals were solubilized by 100 µl acidified SDS solution (10% SDS/0.01 M HCl). Biotek® plate reader measured the absorbance after 14 hours of incubation at 37°C and 5% CO2 at 570 nm. Each experiment was repeated three times, and standard deviation was calculated (±). IC50 was calculated as the concentrations that cause 50% inhibition for cell growth.


## 2.1. Chemistry

### 2.1.1. General

All reagents and solvents were purchased from commercial suppliers and were used without further purification; NMR spectra (1H-NMR and 13C-NMR) were recorded on a JEOL 400 MHz spectrometer. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) were run in either deuterated dimethylsulphoxide (DMSO-d6) or deuterated chloroform (CDCl3). 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.


### 2.1.2. 6-Chloro-4,6-disubstituted s-Triazine Derivatives (2a-i)

6-Chloro-4,6-disubstituted s-triazine derivatives (2a-i) were synthesized following the strategies and methods already reported by our group and others [47–51], as shown in Scheme 1. 6-Hydrazine-4,6-disubstituted s-triazine derivatives were synthesized following the published and reported method by our group [47, 49]. All spectral data were in good agreement with the reported data.Scheme 1
Synthetic route for preparation of s-triazine Schiff-base derivatives.The compounds of4f, 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]. The 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.


### 2.1.3. General Procedure for the Synthesis of Schiff Bases4a-i and 5a-h

A solution of hydroxybenzaldehyde derivatives3a-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). The solvent was evaporated slowly, to provide the corresponding product 4a-i and 5a-h.(1) (E)-2,6-Di-tert-butyl-4-((2-(4,6-di(piperidin-1-yl)-1,3,5-triazin 2-yl)hydrazono)methyl)phenol (4a). Yellow powder in yield 93%; m.p: >250°C; IR (KBr, cm−1): 3637, 3423, 3197, 2960, 2935, 1583, 1570, 1510, 1462;1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.55 (m, 8H, 4CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 4H, 2CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 206.9, 156.6, 136.0, 128.9,124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 494.70 [M + 1]+; Anal. for C28H43N7O; Calcd: C, 68.12; H, 8.78; N, 19.86; Found: C, 68.13; H, 8.79; N, 19.89.(2) (E)-2,6-Di-tert-butyl-4-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4b). Yellow powder in yield 91%; m.p: 180°C; IR (KBr, cm−1): 3086, 3053, 2953, 2885,1598, 1560, 1516, 1463, 1371;1H-NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1H, CH =), 8.10 (brs, 1H, NH), 7.38 (s, 1H, Ph), 7.12 (s, 1H, Ph), 3.70 (brs, 8H, 4CH2), 1.50 (m, 8H, 4CH2), 1.35 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.9, 155.3, 135.1, 129.0, 125.1, 65.9, 44.6, 34.2, 30.9, 30.1LC/MS (ESI): 498.31 [M + 1]+; Anal. for C26H39N7O3; Calcd: C, 62.75; H, 7.90; N, 19.70; Found: C, 62.71; H, 7.91 N, 19.72.(3) (E)-2,6-Di-tert-butyl-4-((2-(4-(diethylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4c). Yellow powder in yield 89%; m.p: 195°C; IR (KBr, cm−1): 3420, 2999, 2960, 2908, 2870, 1624, 1591, 1456;1H-NMR (400 MHz, DMSO-d6): δ 8.60 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.66 (s, 1H, Ph), 7.50 (s, 1H, Ph), 3.82 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.49 (s, 9H, 3CH3), 1.44 (s, 9H, 3CH3), 1.18 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.4, 176.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.1, 12.3; LC/MS (ESI): 484.33 [M + 1]+; Anal. for C26H41N7O2; Calcd: C, 64.57; H, 8.54; N, 20.27; Found: C, 64.57; H, 8.53; N, 20.25.(4) (E)-2,6-Di-tert-butyl-4-((2-(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4d). Yellow powder in yield 80%; m.p: 114°C; IR (KBr, cm−1): 3215, 3045, 2958, 2908, 2866, 1614, 1598, 1544,1463;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 443.27 [M + 1]+; Anal. for C23H34N6O3; Calcd: C, 62.42; H, 7.74; N, 18.99; Found: C, 62.43; H, 7.75; N, 19.01.(5) (E)-2,6-Di-tert-butyl-4-((2-(4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4e). Yellow powder in yield 90%, m.p: >250°C; IR (KBr, cm−1): 3215,3157, 3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463;1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.50 (m, 4H, 2CH2), 1.45 (m, 4H, 2CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 496.33 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 65.43; H, 8.34; N, 19.78; Found: C, 65.43; H, 8.35; N, 19.76.(6) (E)-4-((2-(4-(Benzylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-2,6-di-tert-butylphenol (4f). Yellow powder in yield 88%, m.p: 169°C; IR (KBr, cm−1): 3421, 3116, 2958, 2860, 1618, 1591, 1508, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.37-7.28 (m, 5H, Ph), 7.19 (s, 1H, Ph), 4.23 (s, 2H, CH2Ph), 3.73 (brs, 4H, 2CH2), 1.50 (m, 4H, 2CH2), 1.40 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 65.9, 44.7, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 518.32 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 67.28; H, 7.59; N, 18.94; Found: C, 67.30; H, 7.60; N, 18.95.(7) (E)-2,6-Di-tert-butyl-4-((2-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4g). Yellow powder in yield 87%; m.p: 151°C; IR (KBr, cm−1): 3448, 3215, 3169, 2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.35 (s, 1H, CH =), 8.02 (brs, 1H, NH), 7.50 (s, 1H, Ph), 7.30-7.26 (m, 5H, Ph), 7.20 (s, 1H, Ph), 3.70 (brs, 4H, 2CH2), 1.56 (m, 4H, 2CH2), 1.35 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 66.0, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 504.30 [M + 1]+; Anal. for C28H37N7O2; Calcd: C, 66.77; H, 7.41; N, 19.47; Found: C, 66.76; H, 7.40; N, 19.49.(8) (E)-2,6-Di-tert-butyl-4-((2-(4-methoxy-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4h). Yellow powder in yield 93%; m.p: 124°C; IR (KBr, cm−1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3), 1.35 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 441.29 [M + 1]+; Anal. for C24H36N6O2; Calcd: C, 65.43; H, 8.24; N, 19.07; Found: C, 65.44; H, 8.23; N, 19.10.(9) (E)-2,6-Di-tert-butyl-4-((2-(4-(diethylamino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4i). Yellow powder in yield 94%; m.p: 169°C; IR (KBr, cm−1): 3423, 3197, 2960, 2908, 2858, 1583, 1570, 1510, 1435;1H-NMR (400 MHz, DMSO-d6): δ 8.44 (s, 1H, CH =), 7.95 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.47 (s, 1H, Ph), 3.76 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.49 (s, 9H, 3CH3), 1.44 (s, 9H, 3CH3), 128 (m, 2H, CH2), 1.22 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 199.3, 176.1, 156.2, 142.8, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.0, 12.2; LC/MS (ESI): 482.35 [M + 1]+; Anal. for C27H43N7O; Calcd: C, 67.32; H, 9.00; N, 20.36; Found: C, 67.31; H, 9.00; N, 20.35.(10) (E)-2,4-Di-tert-butyl-6-((2-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5a). Yellow powder in yield 90%; m.p: 217°C; IR (KBr, cm−1): 3637, 3423, 3197, 2960, 2935, 1583, 1570, 1510, 1462;1H-NMR (400 MHz, DMSO-d6): δ 8.03 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.32 (s, 1H, Ph), 7.01 (s, 1H, Ph), 3.79 (brs, 8H, 4CH2), 1.51 (m, 8H, 4CH2), 1.38 (s, 18H, 6CH3), 1.27 (m, 4H, 2CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 206.9, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 494.35 [M + 1]+; Anal. for C28H43N7O; Calcd: C, 68.12; H, 8.78; N, 19.86; Found: C, 68.11; H, 8.78; N, 19.83.(11) (E)-2,4-Di-tert-butyl-6-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5b). Yellow powder in yield 95%; m.p: 188°C; IR (KBr, cm−1): 3086, 3053, 2953, 2885,1598, 1560, 1516, 1463, 1371;1H-NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1H, CH =), 8.10 (brs, 1H, NH), 7.38 (s, 1H, Ph), 7.12 (s, 1H, Ph), 3.70 (brs, 8H, 4CH2), 1.50 (m, 8H, 4CH2), 1.35 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.9, 155.3, 135.1, 129.0, 125.1, 65.9, 44.6, 34.2, 30.9, 30.1; LC/MS (ESI): 498.31 [M + 1]+; Anal. for C26H39N7O3; Calcd: C, 62.75; H, 7.90; N, 19.70; Found: C, 62.75; H, 7.89; N, 19.72.(12) (E)-2,4-Di-tert-butyl-6-((2-(4-(diethylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5c). Yellow powder in yield 87%; m.p: 203°C; IR (KBr, cm−1): 3421, 3116, 2966, 2980, 1647, 1618, 1591, 1508,1499, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.82 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.53 (s, 1H, Ph), 7.54 (s, 1H, Ph), 3.82 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.57 (s, 9H, 3CH3), 1.40 (s, 9H, 3CH3), 1.18 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.4, 176.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.1, 12.3; 13C-NMR (100 MHz, DMSO-d6): δ = ; LC/MS (ESI): 484.33 [M + 1]+; Anal. for C26H41N7O2; Calcd: C, 64.57; H, 8.54; N, 20.27; Found: C, 64.56; H, 8.53; N, 20.24.(13) (E)-2,4-Di-tert-butyl-6-((2-(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5d). Yellow powder in yield 83%; m.p: 195°C; IR (KBr, cm−1): 3215, 3045, 2958, 2908, 2866, 1614, 1598, 1544,1463;1H-NMR (400 MHz, DMSO-d6): δ 7.95 (s, 1H, CH =), 7.78 (brs, 1H, NH), 7.65 (s, 1H, Ph), 7.50 (s, 1H, Ph), 3.92 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 443.27 [M + 1]+; Anal. for C23H34N6O3; Calcd: C, 62.42; H, 7.74; N, 18.99; Found: C, 62.42; H, 7.75; N, 19.22.(14) (E)-2,4-Di-tert-butyl-6-((2-(4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5e). Yellow powder in yield 86%; m.p: 171°C; IR (KBr, cm−1) 3134, 3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463; 1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.50 (m, 4H, 2CH2), 1.45 (m, 4H, 2CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 496.33 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 65.43; H, 8.34; N, 19.78; Found: C, 65.44; H, 8.35; N, 19.97.(15) (E)-2-((2-(4-(Benzylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-4,6-di-tert-butylphenol (5f). Yellow powder in yield 85%; m.p: 152°C; IR (KBr, cm−1): 3421, 3116, 2958, 2860, 1618, 1591, 1508, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.37-7.28 (m, 5H, Ph), 7.19 (s, 1H, Ph), 4.23 (s, 2H, CH2Ph), 3.73 (brs, 4H, 2CH2), 1.50 (m, 4H, 2CH2), 1.40 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 65.9, 44.7, 31.2, 30.0, 25.6, 24.5; 13C-NMR (100 MHz, DMSO-d6): δ = ; LC/MS (ESI): 518.32 [M + 1]+; Anal. For C27H41N7O2; Calcd: C, 67.28; H, 7.59; N, 18.94; Found: C, 67.29; H, 7.60; N, 19.06.(16) (E)-2,4-di-tert-butyl-6-((2-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5g). Yellow powder in yield 88%; m.p: 242°C; IR (KBr, cm−1): 3448, 3215, 3169, 2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.35 (s, 1H, CH =), 8.02 (brs, 1H, NH), 7.50 (s, 1H, Ph), 7.30-7.26 (m, 5H, Ph), 7.20 (s, 1H, Ph), 3.70 (brs, 4H, 2CH2), 1.56 (m, 4H, 2CH2), 1.35 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 66.0, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 504.30 [M + 1]+; Anal. for C28H37N7O2; Calcd: C, 66.77; H, 7.41; N, 19.47; Found: C, 66.77; H, 7.41; N, 19.48.(17) (E)-2,4-Di-tert-butyl-4-((2-(4-methoxy-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5h). Yellow powder in yield 81%; m.p: 155°C; IR (KBr, cm−1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3), 1.35 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 441.29 [M + 1]+; Anal. for C24H36N6O2; Calcd: C, 65.43; H, 8.24; N, 19.07; Found: C, 65.42; H, 8.25; N, 19.20.


## 2.1.1. General

All reagents and solvents were purchased from commercial suppliers and were used without further purification; NMR spectra (1H-NMR and 13C-NMR) were recorded on a JEOL 400 MHz spectrometer. 1H-NMR (400 MHz) and 13C-NMR (100 MHz) were run in either deuterated dimethylsulphoxide (DMSO-d6) or deuterated chloroform (CDCl3). 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.


## 2.1.2. 6-Chloro-4,6-disubstituted s-Triazine Derivatives (2a-i)

6-Chloro-4,6-disubstituted s-triazine derivatives (2a-i) were synthesized following the strategies and methods already reported by our group and others [47–51], as shown in Scheme 1. 6-Hydrazine-4,6-disubstituted s-triazine derivatives were synthesized following the published and reported method by our group [47, 49]. All spectral data were in good agreement with the reported data.Scheme 1
Synthetic route for preparation of s-triazine Schiff-base derivatives.The compounds of4f, 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]. The 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.


## 2.1.3. General Procedure for the Synthesis of Schiff Bases4a-i and 5a-h

A solution of hydroxybenzaldehyde derivatives3a-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). The solvent was evaporated slowly, to provide the corresponding product 4a-i and 5a-h.(1) (E)-2,6-Di-tert-butyl-4-((2-(4,6-di(piperidin-1-yl)-1,3,5-triazin 2-yl)hydrazono)methyl)phenol (4a). Yellow powder in yield 93%; m.p: >250°C; IR (KBr, cm−1): 3637, 3423, 3197, 2960, 2935, 1583, 1570, 1510, 1462;1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.55 (m, 8H, 4CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 4H, 2CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 206.9, 156.6, 136.0, 128.9,124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 494.70 [M + 1]+; Anal. for C28H43N7O; Calcd: C, 68.12; H, 8.78; N, 19.86; Found: C, 68.13; H, 8.79; N, 19.89.(2) (E)-2,6-Di-tert-butyl-4-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4b). Yellow powder in yield 91%; m.p: 180°C; IR (KBr, cm−1): 3086, 3053, 2953, 2885,1598, 1560, 1516, 1463, 1371;1H-NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1H, CH =), 8.10 (brs, 1H, NH), 7.38 (s, 1H, Ph), 7.12 (s, 1H, Ph), 3.70 (brs, 8H, 4CH2), 1.50 (m, 8H, 4CH2), 1.35 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.9, 155.3, 135.1, 129.0, 125.1, 65.9, 44.6, 34.2, 30.9, 30.1LC/MS (ESI): 498.31 [M + 1]+; Anal. for C26H39N7O3; Calcd: C, 62.75; H, 7.90; N, 19.70; Found: C, 62.71; H, 7.91 N, 19.72.(3) (E)-2,6-Di-tert-butyl-4-((2-(4-(diethylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4c). Yellow powder in yield 89%; m.p: 195°C; IR (KBr, cm−1): 3420, 2999, 2960, 2908, 2870, 1624, 1591, 1456;1H-NMR (400 MHz, DMSO-d6): δ 8.60 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.66 (s, 1H, Ph), 7.50 (s, 1H, Ph), 3.82 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.49 (s, 9H, 3CH3), 1.44 (s, 9H, 3CH3), 1.18 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.4, 176.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.1, 12.3; LC/MS (ESI): 484.33 [M + 1]+; Anal. for C26H41N7O2; Calcd: C, 64.57; H, 8.54; N, 20.27; Found: C, 64.57; H, 8.53; N, 20.25.(4) (E)-2,6-Di-tert-butyl-4-((2-(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4d). Yellow powder in yield 80%; m.p: 114°C; IR (KBr, cm−1): 3215, 3045, 2958, 2908, 2866, 1614, 1598, 1544,1463;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 443.27 [M + 1]+; Anal. for C23H34N6O3; Calcd: C, 62.42; H, 7.74; N, 18.99; Found: C, 62.43; H, 7.75; N, 19.01.(5) (E)-2,6-Di-tert-butyl-4-((2-(4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4e). Yellow powder in yield 90%, m.p: >250°C; IR (KBr, cm−1): 3215,3157, 3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463;1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.50 (m, 4H, 2CH2), 1.45 (m, 4H, 2CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 496.33 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 65.43; H, 8.34; N, 19.78; Found: C, 65.43; H, 8.35; N, 19.76.(6) (E)-4-((2-(4-(Benzylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-2,6-di-tert-butylphenol (4f). Yellow powder in yield 88%, m.p: 169°C; IR (KBr, cm−1): 3421, 3116, 2958, 2860, 1618, 1591, 1508, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.37-7.28 (m, 5H, Ph), 7.19 (s, 1H, Ph), 4.23 (s, 2H, CH2Ph), 3.73 (brs, 4H, 2CH2), 1.50 (m, 4H, 2CH2), 1.40 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 65.9, 44.7, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 518.32 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 67.28; H, 7.59; N, 18.94; Found: C, 67.30; H, 7.60; N, 18.95.(7) (E)-2,6-Di-tert-butyl-4-((2-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4g). Yellow powder in yield 87%; m.p: 151°C; IR (KBr, cm−1): 3448, 3215, 3169, 2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.35 (s, 1H, CH =), 8.02 (brs, 1H, NH), 7.50 (s, 1H, Ph), 7.30-7.26 (m, 5H, Ph), 7.20 (s, 1H, Ph), 3.70 (brs, 4H, 2CH2), 1.56 (m, 4H, 2CH2), 1.35 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 66.0, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 504.30 [M + 1]+; Anal. for C28H37N7O2; Calcd: C, 66.77; H, 7.41; N, 19.47; Found: C, 66.76; H, 7.40; N, 19.49.(8) (E)-2,6-Di-tert-butyl-4-((2-(4-methoxy-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4h). Yellow powder in yield 93%; m.p: 124°C; IR (KBr, cm−1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3), 1.35 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 441.29 [M + 1]+; Anal. for C24H36N6O2; Calcd: C, 65.43; H, 8.24; N, 19.07; Found: C, 65.44; H, 8.23; N, 19.10.(9) (E)-2,6-Di-tert-butyl-4-((2-(4-(diethylamino)-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (4i). Yellow powder in yield 94%; m.p: 169°C; IR (KBr, cm−1): 3423, 3197, 2960, 2908, 2858, 1583, 1570, 1510, 1435;1H-NMR (400 MHz, DMSO-d6): δ 8.44 (s, 1H, CH =), 7.95 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.47 (s, 1H, Ph), 3.76 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.49 (s, 9H, 3CH3), 1.44 (s, 9H, 3CH3), 128 (m, 2H, CH2), 1.22 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 199.3, 176.1, 156.2, 142.8, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.0, 12.2; LC/MS (ESI): 482.35 [M + 1]+; Anal. for C27H43N7O; Calcd: C, 67.32; H, 9.00; N, 20.36; Found: C, 67.31; H, 9.00; N, 20.35.(10) (E)-2,4-Di-tert-butyl-6-((2-(4,6-di(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5a). Yellow powder in yield 90%; m.p: 217°C; IR (KBr, cm−1): 3637, 3423, 3197, 2960, 2935, 1583, 1570, 1510, 1462;1H-NMR (400 MHz, DMSO-d6): δ 8.03 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.32 (s, 1H, Ph), 7.01 (s, 1H, Ph), 3.79 (brs, 8H, 4CH2), 1.51 (m, 8H, 4CH2), 1.38 (s, 18H, 6CH3), 1.27 (m, 4H, 2CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 206.9, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 494.35 [M + 1]+; Anal. for C28H43N7O; Calcd: C, 68.12; H, 8.78; N, 19.86; Found: C, 68.11; H, 8.78; N, 19.83.(11) (E)-2,4-Di-tert-butyl-6-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5b). Yellow powder in yield 95%; m.p: 188°C; IR (KBr, cm−1): 3086, 3053, 2953, 2885,1598, 1560, 1516, 1463, 1371;1H-NMR (400 MHz, DMSO-d6): δ 8.25 (s, 1H, CH =), 8.10 (brs, 1H, NH), 7.38 (s, 1H, Ph), 7.12 (s, 1H, Ph), 3.70 (brs, 8H, 4CH2), 1.50 (m, 8H, 4CH2), 1.35 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.9, 155.3, 135.1, 129.0, 125.1, 65.9, 44.6, 34.2, 30.9, 30.1; LC/MS (ESI): 498.31 [M + 1]+; Anal. for C26H39N7O3; Calcd: C, 62.75; H, 7.90; N, 19.70; Found: C, 62.75; H, 7.89; N, 19.72.(12) (E)-2,4-Di-tert-butyl-6-((2-(4-(diethylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5c). Yellow powder in yield 87%; m.p: 203°C; IR (KBr, cm−1): 3421, 3116, 2966, 2980, 1647, 1618, 1591, 1508,1499, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.82 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.53 (s, 1H, Ph), 7.54 (s, 1H, Ph), 3.82 (brs, 4H, 2CH2), 3.72 (t, 4H, J = 22.3 Hz, 2CH2), 3.60 (brs, 4H, 2CH2), 1.57 (s, 9H, 3CH3), 1.40 (s, 9H, 3CH3), 1.18 (m, 6H, 2CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 198.4, 176.5, 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 47.9, 44.7, 34.3, 31.1, 12.3; 13C-NMR (100 MHz, DMSO-d6): δ = ; LC/MS (ESI): 484.33 [M + 1]+; Anal. for C26H41N7O2; Calcd: C, 64.57; H, 8.54; N, 20.27; Found: C, 64.56; H, 8.53; N, 20.24.(13) (E)-2,4-Di-tert-butyl-6-((2-(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5d). Yellow powder in yield 83%; m.p: 195°C; IR (KBr, cm−1): 3215, 3045, 2958, 2908, 2866, 1614, 1598, 1544,1463;1H-NMR (400 MHz, DMSO-d6): δ 7.95 (s, 1H, CH =), 7.78 (brs, 1H, NH), 7.65 (s, 1H, Ph), 7.50 (s, 1H, Ph), 3.92 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 443.27 [M + 1]+; Anal. for C23H34N6O3; Calcd: C, 62.42; H, 7.74; N, 18.99; Found: C, 62.42; H, 7.75; N, 19.22.(14) (E)-2,4-Di-tert-butyl-6-((2-(4-morpholino-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5e). Yellow powder in yield 86%; m.p: 171°C; IR (KBr, cm−1) 3134, 3045, 2958, 2908, 2866, 1614, 1598, 1544, 1463; 1H-NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.46 (s, 1H, Ph), 7.19 (s, 1H, Ph), 3.73 (brs, 8H, 4CH2), 1.50 (m, 4H, 2CH2), 1.45 (m, 4H, 2CH2), 1.38 (s, 18H, 6CH3), 1.34 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 128.9, 124.4, 44.7, 34.3, 30.9, 30.1, 25.8, 24.6; LC/MS (ESI): 496.33 [M + 1]+; Anal. for C27H41N7O2; Calcd: C, 65.43; H, 8.34; N, 19.78; Found: C, 65.44; H, 8.35; N, 19.97.(15) (E)-2-((2-(4-(Benzylamino)-6-morpholino-1,3,5-triazin-2-yl)hydrazono)methyl)-4,6-di-tert-butylphenol (5f). Yellow powder in yield 85%; m.p: 152°C; IR (KBr, cm−1): 3421, 3116, 2958, 2860, 1618, 1591, 1508, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.55 (s, 1H, Ph), 7.37-7.28 (m, 5H, Ph), 7.19 (s, 1H, Ph), 4.23 (s, 2H, CH2Ph), 3.73 (brs, 4H, 2CH2), 1.50 (m, 4H, 2CH2), 1.40 (s, 18H, 6CH3); 13C-NMR (100 MHz, DMSO-d6):δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 65.9, 44.7, 31.2, 30.0, 25.6, 24.5; 13C-NMR (100 MHz, DMSO-d6): δ = ; LC/MS (ESI): 518.32 [M + 1]+; Anal. For C27H41N7O2; Calcd: C, 67.28; H, 7.59; N, 18.94; Found: C, 67.29; H, 7.60; N, 19.06.(16) (E)-2,4-di-tert-butyl-6-((2-(4-morpholino-6-(phenylamino)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5g). Yellow powder in yield 88%; m.p: 242°C; IR (KBr, cm−1): 3448, 3215, 3169, 2953, 2866, 1595, 1570, 1554, 1476, 1435, 1440;1H-NMR (400 MHz, DMSO-d6): δ 8.35 (s, 1H, CH =), 8.02 (brs, 1H, NH), 7.50 (s, 1H, Ph), 7.30-7.26 (m, 5H, Ph), 7.20 (s, 1H, Ph), 3.70 (brs, 4H, 2CH2), 1.56 (m, 4H, 2CH2), 1.35 (s, 18H, 6CH3);13C-NMR (100 MHz, DMSO-d6): δ = 198.7, 156.6, 136.0, 129.5, 128.9, 124.4, 123.2, 66.0, 31.2, 30.0, 25.6, 24.5; LC/MS (ESI): 504.30 [M + 1]+; Anal. for C28H37N7O2; Calcd: C, 66.77; H, 7.41; N, 19.47; Found: C, 66.77; H, 7.41; N, 19.48.(17) (E)-2,4-Di-tert-butyl-4-((2-(4-methoxy-6-(piperidin-1-yl)-1,3,5-triazin-2-yl)hydrazono)methyl)phenol (5h). Yellow powder in yield 81%; m.p: 155°C; IR (KBr, cm−1): 3302, 2958, 2908, 2866, 1614, 1587, 1545, 1504, 1438, 1409;1H-NMR (400 MHz, DMSO-d6): δ 7.94 (s, 1H, CH =), 7.80 (brs, 1H, NH), 7.70 (s, 1H, Ph), 7.52 (s, 1H, Ph), 3.93 (s, 3H, OCH3), 3.83 (brs, 4H, 2CH2), 3.70 (brs, 4H, 2CH2), 1.45 (s, 9H, 3CH3), 1.43 (s, 9H, 3CH3), 1.35 (m, 2H, CH2); 13C-NMR (100 MHz, DMSO-d6): δ = 194.6, 173.5 156.6, 143.9, 136.0, 125.3, 124.4, 67.5, 53.9, 47.9, 34.3, 31.0; LC/MS (ESI): 441.29 [M + 1]+; Anal. for C24H36N6O2; Calcd: C, 65.43; H, 8.24; N, 19.07; Found: C, 65.42; H, 8.25; N, 19.20.


## 2.2. Anticancer Activity

The 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). The cell lines were obtained from VACSERA, holding company for biological products and vaccines, Cairo, Egypt. The cells were grown at 37°C and 10% CO2 in DMEM (Lonza, 12-604F) medium supplemented with 10% fetal bovine serum (FBS, Lonza, Cat. No.14-801E), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Lonza, 17-602E). The viability of the cells were quantified by using MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), which measures the activity of mitochondrial dehydrogenase in the viable cells [54].The cells were seeded in 96-well plates as 5 × 104 cells/mL (100 µl/well). Six serial dilutions of tested compounds were added after overnight incubation of the cells at 37°C and 5% CO2. DMSO was used as a negative control (0.5 %). The 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. The formazan crystals were solubilized by 100 µl acidified SDS solution (10% SDS/0.01 M HCl). Biotek® plate reader measured the absorbance after 14 hours of incubation at 37°C and 5% CO2 at 570 nm. Each experiment was repeated three times, and standard deviation was calculated (±). IC50 was calculated as the concentrations that cause 50% inhibition for cell growth.


## 3. Results and Discussion

### 3.1. Chemistry

The s-triazine Schiff-base derivatives4a-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 Na2CO3 at 0–5°C for 2 h. The second amine was added to the reaction mixture followed by addition of equivalent amount of Na2CO3 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) 6-chloro-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) 6-hydrazino-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).The final products were separated by simple filtration after the precipitation. The 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).Figure 2
ORTEP diagram of the titled compounds4f (a), 5b (b), and 5f (c). Displacement ellipsoids are plotted at the 40% probability level for non-H atoms.
(a)
(b)
(c)In the title compounds, the crystallographic data and refinement information are summarized in Figures S1–S6 and Tables S1–S9 (Supplementary Materials). The asymmetric unit of the three containing is shown in Figure2, 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 compound5b are linked together by one hydrogen bond between N6—H1N6⋯O1. Compound 5f molecules are connected with each other via three hydrogen bonds O2—H2A···O1, N5—H5A···N3, and N6—H6C⋯N1. Finally, molecules of structure 5f arranged in the crystal structure using only one hydrogen bond between N4—H1N4⋯O2.


### 3.2. 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 10µM 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).Figure 3
Cytotoxic activity for4a-i and 5a-h against breast cancer cells (MCF-7).Figure 4
Anticancer activity4a-i and 5a-h against colon cancer cells (HCT-116).Based on the results obtained and showed in Figures3 and 4, we can concluded that 2-hydroxybenzylidene derivatives showed better activity than the analogous 4-hydroxybenzylidene 5a-h derivatives vs. 4a-h derivatives even in the weak activity. This might be due to in the 4-position 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. The 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. The 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. These observations agreed with our previously reported data [44].As noticed from Figure5, the concentration that kills 50% of breast cancer cells was evaluated for the two most active compounds 4a and 5a. Compound 4a showed IC50 of 27 µM (13.3 µg/mL), while 5a was more active at IC50 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).Figure 5
Cytotoxicity of4a and 5a against MCF-7 cells. Cells were treated with DMSO, 4a, and 5a or cisplatin for 48 hours and then the viability was determined by MTT assay.


## 3.1. Chemistry

The s-triazine Schiff-base derivatives4a-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 Na2CO3 at 0–5°C for 2 h. The second amine was added to the reaction mixture followed by addition of equivalent amount of Na2CO3 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) 6-chloro-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) 6-hydrazino-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).The final products were separated by simple filtration after the precipitation. The 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).Figure 2
ORTEP diagram of the titled compounds4f (a), 5b (b), and 5f (c). Displacement ellipsoids are plotted at the 40% probability level for non-H atoms.
(a)
(b)
(c)In the title compounds, the crystallographic data and refinement information are summarized in Figures S1–S6 and Tables S1–S9 (Supplementary Materials). The asymmetric unit of the three containing is shown in Figure2, 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 compound5b are linked together by one hydrogen bond between N6—H1N6⋯O1. Compound 5f molecules are connected with each other via three hydrogen bonds O2—H2A···O1, N5—H5A···N3, and N6—H6C⋯N1. Finally, molecules of structure 5f arranged in the crystal structure using only one hydrogen bond between N4—H1N4⋯O2.


## 3.2. 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 10µM 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).Figure 3
Cytotoxic activity for4a-i and 5a-h against breast cancer cells (MCF-7).Figure 4
Anticancer activity4a-i and 5a-h against colon cancer cells (HCT-116).Based on the results obtained and showed in Figures3 and 4, we can concluded that 2-hydroxybenzylidene derivatives showed better activity than the analogous 4-hydroxybenzylidene 5a-h derivatives vs. 4a-h derivatives even in the weak activity. This might be due to in the 4-position 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. The 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. The 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. These observations agreed with our previously reported data [44].As noticed from Figure5, the concentration that kills 50% of breast cancer cells was evaluated for the two most active compounds 4a and 5a. Compound 4a showed IC50 of 27 µM (13.3 µg/mL), while 5a was more active at IC50 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).Figure 5
Cytotoxicity of4a and 5a against MCF-7 cells. Cells were treated with DMSO, 4a, and 5a or cisplatin for 48 hours and then the viability was determined by MTT assay.


## 4. Conclusions

The new series of s-triazine Schiff-base derivatives were synthesized using the mild and conventional method. The chemical structures of the prepared compounds have been confirmed by different sets of spectroscopic techniques. The 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 effect on the biological activity of the prepared compounds, where 2-hydroxybenzylidene derivatives showed better activity than the analogous 4-hydroxybenzylidene5a-h derivatives vs. 4a-h derivatives even in the weak activity cases. The substituents in the s-triazine ring have affected the activity 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. The concentration that kills 50% of breast cancer cells was evaluated for the two most active compounds 4a and 5a. Compound 5a showed higher activity (IC50 = 17 µM (8.4 µg/mL)) than 4a (IC50 = 27 µM (13.3 µg/mL)). These values, especially those of 5a, are very close to that of the chemotherapeutic drug cisplatin, having IC50 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.



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*Source: 9403908-2019-04-30.xml*