Synthesis, characterization, and antiproliferative and apoptosis inducing effects of novel: S -triazine derivatives

Article abstract of DOI:10.1039/c7nj03348f

In an attempt to design and synthesize a new class of antitumor agents, a mild and eco-friendly protocol for nucleophilic substitution using an s-triazine scaffold, via amine and Schiff base derivatives, has been developed. In order to obtain antitumor activity, all synthesized compounds were screened in vitro for their cytotoxicity against human fibrosarcoma tumor cells (HT-1080) and a cervical cancer cell line (HeLa), for their ability to inhibit the growth of cancer cells. The selected s-triazine analogs (5c, 5d, and 6c) have been preliminarily studied for their reactive oxygen species (ROS) properties, mitochondrial membrane potential (MMP) and apoptosis (AO/EtBr) activity against the HT-1080 cancer cell line. The in vitro anticancer activity analysis has revealed that the synthesized compounds have good/moderate inhibitory activity against the tested cell lines compared to the standard drug. The theoretical study results also provide evidence that the s-triazines scaffolds have been successfully identified as superior p53-MDM2 inhibitors through structure-based design.

Full text of DOI:10.1039/c7nj03348f

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K. Narayanan, H. kamatam, D. Karthikeyan, A. R, L. Chandrasekaran and V. Chidambaranathan, New J.
Chem., 2017, DOI: 10.1039/C7NJ03348F.
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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Synthesis, Characterization, Antiproliferative and Apoptosis
Inducing Effects of Novel s-Triazine Derivatives
Mani Shanmugam a,g,*, Kuppusamy Narayanan b, Kamatam Hari Prasad a,c,*, Dhanapalan
Karthikeyan d, Loganathan Chandrasekaran e, Raji Atchudan f,*, V. Chidambaranathan g,*
aDepartment of Science and Humanities, Institute of Aeronautical Engineering, Dundigal, Hyderabadꢀ500 043,
India.
bDepartment of Chemistry, Excel Engineering College, Komarapalayam, Tamilnadu– 637 303, India
cDepartment of Physics, Pondicherry University, Pondicherry ꢀ 605 014, India.
dDepartment of Chemistry, Arignar Anna College of Arts and Science, Krishnagiri, Tamilnadu– 635 00, India.
eResearch and Development, Gland Pharma Ltd, Dundigal, Hyderabad ꢀ 500 043, India.
fSchool of Chemical Engineering, Yeungnam University, Gyeongsan– 38541, Republic of Korea.
gChemistry Section, FEAT, Annamalai University, Annamalainagar, Tamilnadu– 608 002, India.
Abstract: In an attempt to design and synthesis of new class antitumor agents, the mild and ecoꢀ
friendly protocol for the nucleophilic substitution at sꢀtriazine scaffold via amine and Schiff base
derivatives has been developed. In order to provide the antitumor activity, all the synthesized
compounds were screened in vitro for their cytotoxicity against human fibro sarcoma tumor cells
(HTꢀ1080) and cervical cancer cell line (HeLa) for their ability to inhibit the growth of cancer
cells. The selected sꢀtriazine analogs (5c, 5d, and 6c) have been preliminarily studied for their
reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and apoptosis
(AO/EtBr) activity against HTꢀ1080 cancer cell line. The in vitro anticancer activity analysis has
revealed that the synthesized compounds have good/moderate inhibitory activity against tested
cell lines compared to the standard drug. The theoretical studies result also provide evidence that
the sꢀtriazines scaffolds are successfully identified as superior p53−MDM2 inhibitors by
structureꢀbased design.
Keywords: sꢀTriazine derivatives; Characterization, Docking studies; Cytotoxicity; ROS; MMP;
Apoptosis.

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∗Corresponding authors. E-mail address: baranchem56@gmail.com (V. Chidambaranathan);
mschemdpi@gmail.com (M. Shanmugam); hariprasad.kamatam@gmail.com (K. Hari Prasad);
atchudanr@yu.ac.kr (R. Atchudan)

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1. Introduction
Cancer is one of the leading causes of death all over the world. It accounts for 8.8 million
deaths in 2015 (World Health Organization). Currently, chemotherapy is used as one of the
treatments to lessen the harmful effects of cancer. But severe sideꢀeffects are noticed because of
the administered cancer drug in the normal tissues. Moreover, the drug resistance will impact the
effectiveness of any new drug which is developed for the curing the disease categories is
imperative. In this regard, the approach for the development of a new drug against cancer
infection continues to be the topmost priority for the development of science and fundamental
research. At times, any modification of the lead molecule designed by a chemist to increase its
affinity and selectivity for the target will increase both its therapeutic and undesired side effects.
In recent years, considerable attention has been diverted towards the synthesis of sꢀtriazine
(symmetric triazine) analogous to the hope of discovering new and more effective antisense
compounds.
Obviously, cyanuric chloride (CC) starting material has been chosen for the synthesis of
various sꢀtriazines, since the reactivity depends on temperature and a systematic substitution of
chlorine atoms by various nucleophiles.1 Thus these positions in the triple branching scaffold
could be leveraged to generate a large structural diversity and modifications in drug motifs using
heterocycles can also provide favorable biological properties. Even in the field of modern
medicinal chemistry, 1,3,5ꢀtriazine scaffolds have been studied extensively and have remained
the subject of many reviews.1ꢀ4 sꢀTriazine core is currently, a prominent structural motif in a
wide range of biologically active compounds of natural as well as synthetic origin.5ꢀ18

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Among the various sꢀtriazine derivatives reported, triꢀsubstituted 1,3,5ꢀtriazine
derivatives display several biological activities such as anticancer (Fig. 1) and antiꢀHIV
activities.19, 20 Besides, a fluoro fragment is a ubiquitous structure that occurs in a wide variety of
naturally occurring and synthetic compounds exhibiting various important biological activities
such as anticancer,21 antimicrobial,22 antiviral,23 and anesthetic.24 In addition, Schiff base based
anticancer drugs play the leading role in the treatment of various malignant diseases,25ꢀ30 but the
side effects, toxicity, and drug resistance have limited their wide range of clinical applications.
Last decade, numerous Schiff base based compounds have been synthesized and screened for the
anticancer activities to overcome the shortcoming of Schiff base based chemotherapy.
As part of our continuous efforts to bring out potent bioactive molecules, we hereby
report the synthesis of sꢀtriazine derivatives using mild catalyst, via nucleophilic substitution
reaction and their structures analyzed by using microanalysis and spectral techniques. The titled
compounds (5a-d, 6a-c, 8a-c, 9a-b, 12a-c, and 13a-c) were screened for their cytotoxicity
against cervical cancer cell line (HeLa) and human fibro sarcoma tumor cells (HTꢀ1080) through
MTT assay method. In order to extend the anticancer evaluation,31 preliminary mechanistic
studies by measuring the intracellular ROS levels (DCFHꢀDA), mitochondrial membrane
potential (Rhꢀ123 staining) and apoptotic morphological changes (AO/EtBr staining) were
carried out for selected compounds 5c, 5d, and 6c against human fibrosarcoma tumor cells (HTꢀ
1080).
In cell biology, the growth of cancer cells was arrested by inhibiting the overexpression
of MDM2 (Murine Double Minutes) which demonstrate the significant activation of tumor
suppressor protein p53.32 Activated p53 upꢀregulates the transcription of numerous genes

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involved in cell cycle arrest, DNA repair, senescence, and apoptosis.33 The aberrant interaction
between MDM2 and p53, suppresses the significant activity of p53 in approximately half of all
human cancers.34 Hence, many researchers working on designing small molecules to competitive
with MDM2 to bind active site of the P53 protein, blocking MDM2's interaction with p53.35 This
results in expanded levels of unbound p53 and reactivation of its pathways.
Consequently, for all the synthesized compounds, the pharmacokinetic properties and
molecular docking studies were carried out to determine the binding mode of the ligand in the
active site of a protein and to predict the binding affinity between the ligand and protein. The
results provide an evidence that the synthesized morpholinoꢀsꢀtriazines (5a-d, 6a-c, 8a-c, 9a-b,
12a-c, and 13a-c) exhibit potent and selective inhibitors of the MDM2ꢀp53.
2. Experimental
2.1. General Characterization techniques
Melting points (mp) were recorded on open capillary melting point apparatus and are
uncorrected. IR spectra were recorded in AVATAR 330 FTꢀIR Thermo Nicolet spectrometer
(range 4000ꢀ400 cmꢀ1) as KBr pellets. 1H NMR spectra were recorded with on a Bruker AMXꢀ
400 spectrometer operating at 400 MHz spectrometer measured at room temperature, using TMS
as an internal standard. 13C NMR spectra were recorded on the same instrument at 100 MHz and
are referenced using the central line of the solvent signal (CDCl3, DMSOꢀd6 septet at δ= 39.5
ppm). MS spectra were recorded with JOEL GCMATE II instrument. Elemental analyses (C, H,
and N) were performed with a Perkin Elmer 2400 Series II CHN Analyzer. Thin layer
chromatography was carried out on (Fluka) Silica Gel. All the chromatographic purifications
were performed with silica gel 60 (100ꢀ200 or 200ꢀ400 mesh), whereas all TLC (Silica gel) was

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performed on silica gel coated (Merk Kiesel 60 GFꢀ254, 0.2 mm thickness) sheets. All reagents
and solvents commercially obtained (SigmaꢀAldrich®, Himedia®) and were used directly without
further purification.
2.2. Pharmacokinetic Properties prediction
Pharmacokinetic properties of the synthesized compounds (5a-d, 6a-c, 8a-c, 9a-b, 12a-c,
and 13a-c) were studied using Qikprop module from Schrödinger software.36 The drugꢀlike
properties of the compounds were calculated in accordance with Lipinski’s rule of five.37
2.3. Docking Studies
The active site of the protein was found based on the coꢀcrystal structure and their critical
amino acids are carefully chosen for our docking study by existing crystal structure. The 3D
coordinates of the crystallographic structure of p53 protein complex (PDB ID: 4OGN) were
retrieved from Protein Data Bank (www.rscb.com).38 The protein complex was preꢀprocessed
and prepared by protein preparation wizard in Maestro of Schrödinger software.39 Addition of
hydrogens and removal of water molecules beyond 5 Å from the active site of the protein using
review and modify tool. The reviewed protein has been optimized and minimized using refine
tool. Force field OPLSꢀ2005 used to optimize and minimize the protein until the root mean
square deviation (RMSD) reached the value of 0.3 Å.40 The 2D structures of compounds 5a-d,
6a-c, 8a-c, 9a-b, 12a-c, and 13a-c were imported from the project table and the structures were
minimized and geometrically refined using Ligprep module.41 Conformers were generated using
torsional search method with distanceꢀdependent dielectric solvation treatment and OPLS–2005
force field. The extra precision (XP) mode of docking was used to find the best fit molecules in
the active site of p53 protein using Glide application of Schrödinger software suite.42

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2.4. Biological Studies
2.4.1. MTT Assay: Cell Growth Inhibition Assay
Human cervical cancer cell line (HeLa) and human fibrosarcoma tumor cells (HTꢀ1080)
obtained from NCCS (National Centre for Cell Science), Pune, India, were used for the cell
proliferation. The cytotoxic effect of the synthesized compounds 5a-d, 6a-c, 8a-c, 9a-b, 12a-c,
and 13a-c on HTꢀ1080 and HeLa cancer cell lines were evaluated through MTT dye reduction
assay as described earlier. A stock solution of every hybrid (5a-d, 6a-c, 8a-c, 9a-b, 12a-c, and
13a-c in 1 mg/ml) was prepared in 0.5% dimethyl sulphoxide (DMSO) (w/v) and stored at 4 °C.
Further dilution was made in culture media to obtain the desired concentrations. The final
concentrations of hybrids in the culture medium were not more than 0.01% (v/v). DMSO, 0.01%
was used as the same control. Cells were treated with different concentrations (10, 20, 30, 40, 50
and 60 ꢁg/ml) of one hybrid after another and the cytotoxicity was observed by MTT assay. The
cells (HeLa) and HTꢀ1080were feed at a density of 6000 cells/well in a 96ꢀwell plate (Corning,
Sigma, USA) and allowed to attach overnight. The cells were treated in triplicate with great
concentration of compounds at 37 ºC for 24 h. After 24 h, the cells were treated with various
concentrations of the compounds 5a-d, 6a-c, 8a-c, 9a-b, 12a-c, and 13a-c in the range of 10ꢀ60
ꢁg/ml and incubated for 24 h and 48 has indicated. After 24 h plates were taken from the CO2
incubator and added 10 µl of MTT (5 mg/ml) to keep CO2 incubator for 4 h. The purple
formazan crystals formed after 4 h were dissolved in equal volume of DMSO after aspirating the
MTT and incubated for further 10 minutes. The mitochondrial dehydrogenase in viable cells that
reduces MTT to blue formazan product was measured at 570 nm (measurement) and 630 nm
(reference) using a 96 wells plate reader (BioꢀRad, Hercules, CA, USA). Data were collected

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from six independent experiments and used to calculate the respective means. The cytotoxicity
and viability of cells has been calculated by following formula;
ꢀꢁꢂꢃ ꢄꢅꢃꢆꢇꢈꢉ ꢊꢁꢋꢂꢆꢃꢌ
% of Cytotoxicity =
x 100
ꢍꢄꢋꢃꢎꢄꢉ ꢄꢅꢃꢆꢇꢈꢉ ꢊꢁꢋꢂꢆꢃꢌ
The results were also expressed as IC50 (the compound concentration required for 50% growth
inhibition of cancer cells) values which were calculated by the logit method.
2.4.2. Measurement of intracellular ROS in cells by spectrofluorometric method
ROS was measured by using a nonꢀfluorescent probe, 2,7ꢀdiacetyl dichlorofluorescein
(DCFHꢀDH) that can penetrate into the intracellular matrix of cells where it is oxidized by ROS to
fluorescent dichlorofluorescein (DCF).43 Briefly, an aliquot of the above mentioned isolated cells
(8 x 106) was made up to a final volume of 2 mL in normal buffered saline (pH 7.4). A one mL
aliquot of cells was taken to which 100 µL DCFHꢀDA (10 ꢁM) was added and incubated at 37°C
for 30 min. Fluorescence measurements were made using a spectrofluorometer (Shimadzu RFꢀ
5301 PC spectrometer) with excitation and emission filters set at 485±10 and 530±12.5 nm,
respectively. Results were expressed as percentage increase in fluorescent intensity.
2.4.3. Alteration in mitochondrial membrane potential
After incubation with different compounds for 24 h, fluorescent dye Rhodamineꢀ123 (10
ꢁg/mL) was added to the cells which were then kept in an incubator for 30 min. Then the cells
were washed with PBS and viewed under the fluorescence microscope using the blue filter. Rhꢀ
123 is a lipophilic cationic dye, highly specific for mitochondria. Orangeꢀred fluorescence
indicated polarized mitochondria and depolarized mitochondria emitted green fluorescence.

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2.4.4. Apoptotic morphological changes by acridine orange-ethidium bromide dual staining
method
Acridine orange/Ethidium Bromide (AO/EtBr) staining was used to visualize apoptotic
cells.44 The control, cancer cells were seeded in 6ꢀwell plates (3 x 104/well) and incubated in a
CO2 incubator for 24 h. The cells were fixed in methanol and glacial acetic acid (3:1) mixture for
30 min at room temperature. The cells were washed with PBS and stained with 1:1 ratio of
AO/EtBr. Stained cells were immediately washed with PBS and viewed under a fluorescence
microscope (Nikon, Eclipse TS100, Japan) with a magnification of 40X. The number of cells
showing features of apoptosis was counted as a function of the total number of cells present in
the field.
2.5. Synthetic procedure of s-triazine derivatives.
2.5.1. General procedure for synthesis of Schiff base compounds (4a-d and 7a-c).
Equimolar quantities (0.5 equiv) of 4ꢀhydroxy benzaldehyde as well as 3ꢀfluoroꢀ
salicylaldehyde, followed by the addition of various amines were dissolved in methanol (10 mL)
in the presence of ionic liquid (0.5 equiv) (1ꢀButylꢀ3ꢀmethylimidazolium bis(trifluoroꢀ
methylsulfonyl) imide). The reaction mixture stirred at room temperature for an hour and
°
refluxed for 4ꢀ6 hours at 50 C. The progress of the reaction was monitored by TLC. After
completion, the reaction mixture was cooled to room temperature, the solid product formed was
filtered, washed three times with water and methanol (2:1) mixture and dried. The crude was
further purified by column chromatography using hexane and ethyl acetate (9:2) mixture as
eluent (Scheme 1).45

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2.5.2. General procedure for the synthesis of mono-substituted morpholino based
s-triazine compound (3)
4ꢀ(4,6ꢀdichloroꢀ1,3,5ꢀtriazinꢀ2ꢀyl) morpholine (3) was prepared according to a literature
procedure.46
2.5.3. General procedure for the synthesis of bis-substituted morpholino based s-triazine
Schiff base compounds (5a-d).
Ionic liquid BMIM [Tf2N] (l equiv), 4ꢀ(4,6ꢀdichloroꢀ1,3,5ꢀtriazinꢀ2ꢀyl) morpholine (3) (1
equiv) and Schiff base (4a) (1 equiv) were taken together in a doubleꢀnecked roundꢀbottomed
flask fitted with condenser (Scheme 1 & Table 1). To this, 1 equiv KOH and dry THF/Acetone
(1:2, 10 mL) were added and the reaction mixture was refluxed at 50 °C for 9ꢀ12 h. The progress
of the reaction was monitored by TLC. Upon completion of the reaction, the mixture was
allowed to cool to room temperature, washed with water and chloroform (3:20 mL). Purification
of the product was done by column chromatography using silica gel (60ꢀ120 mesh) and
benzene/ethyl acetate (5:1) as eluent.
(E)-N-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)oxy)benzylidene)-6-ethoxybenzo[d]
thiazol 2-amine (5a)
Yellowish green solid, yield: 95%. Mp: 272ꢀ274ºC; IR (KBr, cmꢀ1) 2960, 2852, 1578,
1
1361, 798. H NMR (DMSOꢀd6, 400 MHz) δ (ppm): 1.32 (t, 3H, J = 6.0 Hz, CH3), 3.64 (s, 4H,
−O−CH2), 3.65 (s, 2H, CH2), 3.74 (t, 4H, J = 6.4 Hz, −N−CH2), 6.96 (d, 1H, J = 12.0 Hz, =CH),
6.97 (d, 2H, J = 8.4 Hz, ArH), 7.36 (d, 1H, J = 8.8 Hz, ArH), 7.45 (s, 1H, ArH), 7.75 (d, 2H, J =
13
8.0 Hz, ArH), 9.78 (s, 1H, −N=CH). C NMR (DMSOꢀd6, 100 MHz) δ 14.5, 44.2, 44.6, 63.7,
65.4, 65.7, 107.9, 114.9, 115.2, 115.8, 125.2, 128.3, 132.7, 153.3, 155.0, 163.3, 163.7, 168.1,

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168.7, 169.1, 190.9. Calc. for C23H21ClN6O3S (%): C, 55.59; H, 4.26; N, 16.91. Found: C, 55.50;
H, 4.31; N, 16.95.
(E)-N-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)oxy)benzylidene)aniline (5b)
ꢀ1
º
Light yellow solid, yield: 95%. Mp: 196ꢀ198 C; IR (KBr, cm ) 2967, 2852, 1580, 1386,
1
1361, 798, 759. H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.60 (s, 4H, −O−CH2), 3.65 (s, 4H,
−N−CH2), 6.92 (d, 2H, J = 8.0 Hz, ArH), 7.22 (d, 2H, J = 7.6 Hz, ArH), 7.20 (t, 1H, J = 11.2 Hz,
13
ArH), 7.39 (t, 2H, J = 7.6 Hz, ArH), 7.81 (d, 2H, J = 8.0 Hz, ArH), 8.48 (s, 1H, −N=CH). C
NMR (DMSOꢀd6, 100 MHz) δ 43.4, 65.6, 115.8, 120.8, 125.4, 129.1, 130.9, 132.0, 151.32,
160.1, 160.9, 163.3, 163.7 190.0. Calc. for C20H18ClN5O2 (%): C, 60.68; H, 4.58; N, 17.69,
Found: C, 60.71; H, 4.54; N, 17.67.
(E)-N-(4-(4-chloro-6-morpholino-1,3,5-triazin-2-yloxy)benzylidene)naphthalen-1-amine
(5c).
Yellow solid, yield: 90%. Mp: 212ꢀ214 ºC; IR (KBr, cmꢀ1) 3057, 2926, 1602, 1581, 1380,
793.1H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.61 (s, 4H, −O−CH2), 3.65 (s, 4H, −N−CH2) 6.96
(d, 2H, J = 2 Hz, ArH), 7.18 (t, 1H, J = 6.8 Hz, ArH), 7.39 (d, 1H, J = 7.2 Hz, ArH), 7.53 (s, 2H,
ArH), 7.76 (d, 2H, J = 6.4 Hz, ArH), 7.91 (d, 2H, J = 7.2 Hz), 8.29 (d, 1H, J = 6.8 Hz), 8.55 (s,
13
1H, −N=CH). C NMR (DMSOꢀd6, 100 MHz) δ 43.4, 65.7, 113.7, 115.7, 122.0, 125.3, 125.6,
126.2, 126.3, 127.4, 127.5, 128.0, 128.3, 130.9, 132.0, 160.4, 160.9, 163.3, 163.7, 190.9. Calc.
for C24H20ClN5O2 (%): C, 64.65; H, 4.52; N, 15.71; Found: C, 64.63; H, 4.52; N, 15.65.

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(E)-N-(4-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)oxy)benzylidene)1phenylmethanamine
(5d).
Light yellowish white, yield: 93%. Mp: 162ꢀ164 ºC; IR (KBr, cmꢀ1) 2965, 2844, 1586,
1366, 799.1H NMR (CDCl3, 400 MHz) (ppm): 3.76 (t, 4H, J = 9.6 Hz, ꢀOꢀCH2), 3.87 (t, 4H, J =
4.4 Hz, −N−CH2), 4.75 (s, 2H, −CH2), 6.86 (d, 2H, J = 8.0 Hz, ArH), 7.24 (t, 1H, J = 4.4 Hz,
ArH), 7.31 (t, 2H, J = 4.4 Hz, ArH), 7.31 (d, 2H, J = 4.4 Hz, ArH), 7.61 (d, 2H, J = 8.0 Hz,
ArH), 8.29 (s, 1H, −N=CH).13C NMR (DMSOꢀd6, 100 MHz) δ: 43.6, 44.5, 64.4, 66.3, 115.6,
126.8, 127.8, 128.7, 133.3, 139.3, 160.1, 164.3, 169.3, 190.7. Calc. for C21H20ClN5O2 (%): C,
61.54; H, 4.92; N, 17.09; Found: C, 61.44; H, 4.96; N, 17.06.
2.5.4. General procedure for synthesis of tri-substituted morpholine based s-triazine
Schiff base compounds (6a-c).
Ionic liquid BMIM [Tf2N] (l equiv), (E)ꢀNꢀ(4ꢀ((4ꢀchloroꢀ6ꢀmorpholinoꢀ1,3,5ꢀtriazinꢀ2ꢀ
yl)oxy)benzylidene)ꢀ6ꢀethoxybenzo[d]thiazol 2ꢀamine (5a) (1 equiv) and morpholine (2) (1
equiv) were taken together in a two necked round bottomed flask fitted with condenser
(Scheme 1). To this, 1 equiv KOH and dry THF/Acetone (1:2, 15 mL) were added and the
0
reaction mixture was refluxed at 50 C for 2ꢀ3 h. The progress of the reaction was monitored by
TLC. Upon completion of the reaction, the mixture was allowed to cool to room temperature,
washed with water and chloroform (3:20 mL). Purification of the product was done by column
chromatography using silica gel (60ꢀ120 mesh) and benzene/ethyl acetate (5:1) as eluent.

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(E)-N-(4-((4,6-dimorpholino-1,3,5-triazin-2-yl)oxy)benzylidene)-6-ethoxybenzo[d]thiazol-2-
amine (6a).
Green solid, yield: 90%. Mp: >250 ºC; IR (KBr, cmꢀ1) 2969, 2852, 1577, 1361, 798.1H
NMR (DMSOꢀd6, 400 MHz) (ppm): 1.29 (t, 3H, J = 8.0 Hz, CH3), 3.72 (q, 2H, CH2), 3.73 (s,
8H, J = 4.8 Hz, −O−CH2), 3.98 (t, 8H, J = 6.4 Hz, −N−CH2), 6.82 (d, 1H, J = 8.8 Hz, =CH), 6.92
(d, 2H, J = 8.4 Hz, ArH), 7.24 (d, 1H, J = 10.0 Hz, ArH), 7.29 (s, 1H, ArH), 7.75 (d, 2H, J = 8.4
Hz, Ar H), 9.76 (s, 1H, −N=CH). 13C NMR (DMSOꢀd6, 100 MHz) δ 14.6, 43.4, 44.1, 63.5, 65.3,
65.7, 106.2, 113.5, 115.7, 117.7, 128.3, 131.2, 132.1, 153.6, 163.2, 163.4 (−N=CH), 163.7
(−C−NT), 165.0, 169.1, 191.0 (−O−CT). Calc. for C27H29N7O4S (%): C, 59.22; H, 5.34; N,
17.90; found: C, 59.27; H, 5.31; N, 17.93.
(E)-N-(4-((4,6-dimorpholino-1,3,5-triazin-2-yl)oxy)benzylidene)aniline (6b).
Light white solid, yield: 98%. Mp: 216ꢀ218 ºC; IR (KBr, cmꢀ1) 2999, 2967, 2852, 1581,
1
1361, 798. H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.54 (s, 8H, ꢀOꢀCH2), 3.65 (s, 8H, ꢀNꢀCH2),
7.12 (d, 2H, ArH), 7.22 (t, 1H, ArH), 7.26 (d, 2H, ArH), 7.35 (d, 2H, ArH), 7.93 (d, 2H, ArH),
13
8.49 (s, 1H, −N=CH). C NMR (DMSOꢀd6, 100 MHz) δ 43.2, 43.5, 65.7, 66.0, 120.3, 121.6,
122.0, 122.0, 128.7, 129.0, 129.4, 130.2, 130.6, 156.0, 158.6, 190.3. Calc. for C24H26N6O3 (%):
C, 64.56; H, 5.87; N, 18.82; Found: C, 64.61; H, 5.84; N, 18.86.
(E)-N-(4-((4,6-dimorpholino-1,3,5-triazin-2-yl)oxy)benzylidene)naphthalen-1-amine (6c).
Light yellow, yield: 90%. Mp: 262ꢀ264 ºC; IR (KBr, cmꢀ1) 3044, 2970, 2920, 1602, 1568,
1
1384, 793. H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.64 (t, 8H, J = 3.2 Hz, −O−CH2), 3.73 (s,
8H, ꢀNꢀCH2), 6.94 (d, 2H, J = 7.6 Hz, ArH), 7.52 (t, 2H, J = 5.6 Hz, ArH), 7.67 (d, 1H, J = 7.2
Hz, ArH), 7.74 (d, 2H, J = 8 Hz ArH), 7.79 (d, 1H, J = 7.6 Hz, Ar H), 8.01 (d, 2H, J = 9.6 Hz),

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13
8.10 (d, 1H, J = 8.0 Hz, ArH), 9.77 (s, 1H, −N=CH). C NMR (DMSOꢀd6, 100 MHz) δ 43.4,
44.1, 65.4, 65.5, 115.8, 122.0, 123.0, 125.8, 125.9, 126.6, 127.0, 127.8, 127.9, 128.2, 128.4,
131.9, 133.3, 133.6, 163.3, 163.42, 169.1, 190.8. Calc. for C28H28N6O3 (%): C, 67.73; H, 5.68;
N, 16.92; Found: C, 67.66; H, 5.74; N, 16.90.
2.5.5. General synthetic procedure for compounds 8a-c
Synthetic procedure for compounds 8a-c is similar to the general procedure for synthesis of 5a-d
(E)-N-(2-(4-chloro-6-morpholino-1,3,5-triazin-2-yloxy)-3-fluorobenzylidene) aniline (8a).
Red solid, yield: 90%. Mp: 210ꢀ212 ºC; IR (KBr, cmꢀ1) 3055, 2964, 1579, 1362, 837, 785,
729.1H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.61 (s, 4H, ꢀOꢀCH2), 3.68 (s, 4H, ꢀNꢀCH2), 6.97 (d,
1H, J = 4.4 Hz), 7.35 (d, 2H, J = 7.2 Hz, ArH), 7.41 (d, 2H, J = 10.4 Hz, ArH), 7.47 (s, 1H,
ArH), 7.47 (s, 1H, ArH), 7.47 (s, 1H, ArH), 9.05 (s, 1H, CH=N−). 13C NMR (DMSOꢀd6, 100
MHz) δ 43.2, 65.4, 118.3, 119.1, 121.0, 121.4, 121.9, 126.0, 127.8, 129.4, 148.6, 149.2, 147.1/
151.6 (ꢀCꢀF), 163.2, 163.7, 190.9. Calc. for C20H17ClFN5O2 (%): C, 58.05; H, 4.14; N, 16.92;
Found: C, 58.01; H, 4.17; N, 16.89.
(E)-N-(2-(4-chloro-6-morpholino-1,3,5-triazin-2-yloxy)-3-fluorobenzylidene)naphthalen-
1amine (8b).
Yellow solid, yield: 87%. Mp: 242ꢀ244 ºC; IR (KBr, cmꢀ1) 3055, 2967, 1584, 1362, 802,
779.1H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.73 (t, 4H, J = 10.0 Hz, ꢀOꢀCH2), 3.85 (t, 4H, J =
4.4 Hz, −N−CH2), 6.92 (d, 1H, J = 3.2 Hz, ArH), 7.24 (t, 2H, J = 7.6 Hz, ArH), 7.29 (t, 1H, J =
7.6 Hz, ArH), 7.48 (d, 1H, J = 8.0 Hz, ArH), 7.56 (d, 2H, Hz, ArH), 7.80 (d, 1H, J = 8.0 Hz,
ArH), 7.88 (t, 1H, J = 6.0 Hz), 8.22 (d, 1H, J = 3.5 Hz), 8.76 (s, 1H, −N=CH). 13C NMR

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(CDCl3/DMSOꢀd6, 100 MHz) δ 43.6, 44.2, 66.3, 66.4, 114.0, 118.5, 119.2, 122.7, 125.8, 126.5,
126.6, 127.3, 127.3, 127.9, 128.2, 133.0, 145.0, 143.3, 149.8/152.3 (−C−F), 163.0, 163.8, 164.2,
170.0, 189.0. Calc. for C24H19ClFN5O2 (%): C, 62.14; H, 4.13, N, 15.10. found: C, 62.22; H,
4.15, N, 15.
(E)-N-(2-((4-chloro-6-morpholino-1,3,5-triazin-2-yl)oxy)-3-fluorobenzylidene)-1-
phenylmethanamine (8c).
Light yellow solid, yield: 90%. Mp: 202ꢀ204 ºC; IR (KBr, cmꢀ1) 2990, 2859, 1582, 1367,
786. 1H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.66 (t, 4H, J = 11.2 Hz, −O−CH2), 3.73 (t, 4H, J =
4.0 Hz, −N−CH2), 4.84 (s, 2H, −N−CH2), 6.79 (d, 1H, J = 4.0 Hz, ArH), 7.27 (t, 1H, J = 6.4 Hz,
ArH), 7.29 (t, 2H, J = 6.4 Hz, ArH), 7.37 (d, 2H, Hz, ArH), 7.40 (t, 1H, Ar H), 7.42 (d, 1H, J=
13
6.4 Hz, ArH), 8.77 (s, 1H, −N=CH). C NMR (DMSOꢀd6, 100 MHz) δ 43.4, 44.2, 60.3, 65.6,
65.7, 116.8, 118.6, 119.8, 127.3, 127.8, 128.6, 137.9, 149.8/152.2 (ꢀCꢀF), 163.4, 163.7, 166.4,
168.7, 169.1, 190.1. Calc. for C21H19ClFN5O2 (%): C, 58.95; H, 4.48; N, 16.37; Found: C, 58.91;
H, 4.54; N, 16.32.
2.5.6. General synthetic procedure for compounds 9a-b.
Synthetic procedure for compounds 9a-b is similar to the general procedure for synthesis
of 6a-c. (Scheme 1)
(E)-N-(2-(4,6-dimorpholino-1,3,5-triazin-2-yloxy)-3-fluorobenzylidene) aniline (9a).
Orange solid, yield: 95%. Mp: 156ꢀ158 ºC; IR (KBr, cmꢀ1) 3060, 2970, 2920, 1580, 1361,
795, 732.1H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.61 (s, 8H, −O−CH2), 3.74 (s, 8H, −N−CH2),
6.96 (d, 1H, J = 4.4 Hz), 7.34 (t, 1H, J = 8.4 Hz, ArH), 7.40 (d, 1H, J = 8.4 Hz, ArH), 7.47 (s,

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2H, ArH), 7.48 (s, 2H, ArH), 7.64 (t, 1H, ArH), 9.04 (s, 1H, CH=N−). Calc. for C24H25FN6O3
(%): C, 62.06; H, 5.42; N, 18.09; Found: C, 61.96; H, 5.51; N, 18.16.
(E)-N-(2-((4,6-dimorpholino-1,3,5-triazin-2-yl)oxy)-3-fluorobenzylidene)naphthalen-1-
amine (9b).
Orange red solid, yield: 91%. Mp: 265ꢀ267 ºC; IR (KBr, cmꢀ1) 3061, 2968, 1584, 1362,
802, 779.1H NMR (DMSOꢀd6, 400 MHz) δ: 1H NMR (DMSOꢀd6, 400 MHz) (ppm): 3.61 (t, 8H,
J = 6.8 Hz, −O−CH2), 3.73 (t, 8H, J = 3.6 Hz, −N−CH2), 6.96 (d, 1H, J = 4.4 Hz, ArH), 7.50 (t,
2H, J = 7.6 Hz, ArH), 7.59 (t, 3H, J = 7.6 Hz, ArH), 7.78 (d, 1H, J = 8.4 Hz, ArH), 7.91 (d, 1H,
13
J= 8.4 Hz, ArH), 7.99 (d, 1H, ArH), 8.16 (d, 1H, J = 7.6 Hz), 9.10 (s, 1H, −N=CH). C NMR
(CDCl3/DMSOꢀd6, 100 MHz) δ 43.4, 44.1, 65.6, 65.7, 114.6, 118.8, 119.5, 121.6, 122.2, 124.4,
126.1, 126.6, 127.7, 128.0, 133.5, 144.7, 148.6, 149.3/151.7 (ꢀCꢀF), 163.4, 163.7, 163.8, 168.7,
169.1, 190.9. Calc. for C28H27FN6O3 (%): C, 65.36; H, 5.29; N, 16.33; Found: C, 65.32; H, 5.35;
N, 16.26.
2.5.7. General procedure for synthesis of N-(4,6-dichloro-1,3,5-triazin-2-yl)-6-
ethoxybenzo[d] thiazol-2-amine (11).
KOH (1 equiv) and ionic liquid (1 equiv) was added to a solution of cyanuric chloride 1
(1 equiv) and 6ꢀethoxybenzo[d]thiazolꢀ2ꢀamine (1 equiv) in dry THF (15 mL) at room
°
temperature. The mixture was reflux at 50 C for 16 h. The resulting residue washed with THF
and water (1:5) to afford the pure compounds (11) (Scheme 2).
Green solid.1H NMR (DMSOꢀd6, 400 MHz) (ppm): 1.30 (s, 3H, CH3), 2.94 (s, 2H, ꢀCH2)
7.01 (s, 1H, ArH), 7.43 (d, 1H, J= 6.4 Hz, ArH), 7.52 (s, 1H, ArH), 10.02 (s, 1H, ꢀNH). 13C

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NMR (DMSOꢀd6, 100 MHz) δ 14.7, 63.4, 106.1, 113.3, 118.0, 131.8, 146.6, 153.4, 155.5, 159.8,
164.7; HRMS (m/z): 342.200.
2.5.8. General procedure for synthesis of mono-substituted amino based s-triazine Schiff
base compounds (12a-c).
Ionic liquid BMIM [Tf2N] (l equiv), (1 equiv) was added to a solution of Nꢀ(4,6ꢀdichloroꢀ
1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ6ꢀethoxybenzo[d]thiazolꢀ2ꢀamine (11) (1 equiv) and Schiff base 4a (1 equiv).
The reaction mixture was taken in a doubleꢀnecked roundꢀbottomed flask fitted with a reflux
condenser (Scheme 2 & Table 2). To this, 1 equiv KOH and dry Acetone/THF (2:1, 15 mL)
0
were added and the reaction mixture was refluxed at 50 C for 13ꢀ15 h. The progress of the
reaction was monitored by TLC. Upon completion of the reaction, the mixture was allowed to
cool to room temperature. The resulting residue washed with THF and water (1:5) to afford the
pure compounds (12aꢀc).
(E)-N-(4-((4-chloro-6-((6-ethoxybenzo[d]thiazol-2-yl)amino)-1,3,5-triazin-2yl)oxy)
benzylidene)-6-ethoxybenzo[d]thiazol-2-amine (12a).
Green solid, yield: 83%. Mp: 292ꢀ294 °C; IR (KBr, cmꢀ1) 3260, 3216, 2975, 1603, 1570,
1
1389, 1348, 798. H NMR (DMSOꢀd6, 400 MHz) (ppm): 1.34 (s, 6H, −CH2−CH3), 4.0 (s, 4H, ꢀ
O−CH2), 6.86 (s, 2H, ArH), 6.94 (s, 2H, ArH), 7.74 (s, 2H, ArH), 7.72 (s, 2H, ArH), 7.72 (s, 2H,
13
ArH), 9.78 (s, 1H, −N=CH), 10.78 (s, 1H, −NH). C NMR (DMSOꢀd6, 100 MHz) δ 14.6, 63.5,
106.7, 114.6, 115.7, 116.5, 122.2, 123.5, 128.2, 128.8, 131.8, 140.2, 149.8, 154.3, 163.3, 166.2,
166.2, 190.5. Calc. for C28H22ClN7O3S2 (%): C, 55.67; H, 3.67; N, 16.23; found: C, 55.63; H,
3.71; N, 16.25.

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(E)-N-(4-chloro-6-(4-((phenylimino)methyl)phenoxy)-1,3,5-triazin-2-yl)-6-ethoxybenzo
[d]thiazol-2-amine (12b).
Yellowish green solid, yield: 93%. Mp: 268ꢀ270 ºC; IR (KBr, cmꢀ1) 3216, 2978, 1600,
1573, 1365, 753. 1H NMR (DMSOꢀd6, 400 MHz) (ppm): 1.28 (s, 3H, −CH2−CH3), 3.97 (s, 2H, ꢀ
OꢀCH2), 6.96 (s, 2H, ArH), 7.27 (s, 2H, ArH), 7.33 (s, 1H, ArH), 7.40 (s, 2H, ArH), 7.40 (s, 1H,
ArH), 7.40 (s, 1H, ArH), 7.74 (d, 2H, J = 6.4 Hz, ArH), 7.98 (s, 1H, ArH), 9.76 (s, 1H, CH=N−),
13
10.78 (s, 1H, ꢀNH). C NMR (DMSOꢀd6, 100 MHz) δ 14.5, 63.6, 107.2, 114.5, 115.8, 120.8,
121.7, 126.0, 126.4, 127.4, 128.3, 129.2, 129.5, 132.0, 132.5, 149.9, 154.7, 161.1, 162.5, 163.3,
190.9. HRMS (m/z): 502.1453. Calc. for C25H19ClN6O2S (%): C, 59.70; H, 3.81; N, 16.71;
Found: C, 59.71; H, 3.76; N, 16.78.
(E)-N-(4-chloro-6-(4-((naphthalen-1-ylimino)methyl)phenoxy)-1,3,5-triazin-2-yl)-6-
ethoxybenzo[d]thiazol-2-amine(12c).
Greenish yellow solid, yield: 90%. Mp: > 300 ºC; IR (KBr, cmꢀ1) 3222, 3046, 2978, 1602,
1582, 1385, 793.1H NMR (DMSOꢀd6, 400 MHz) (ppm): 1.29 (s, 3H, −CH2−CH3), 3.95 (s, 2H,
−O−CH2), 6.92 (s, 2H, ArH), 7.09 (s, 1H, ArH), 7.21 (s, 1H, ArH), 7.49 (s, 2H, ArH), 7.49 (s,
1H, ArH), 7.69 (s, 1H, ArH), 7.86 (s, 2H, ArH), 7.86 (s, 1H ArH), 7.86 (s, 1H, ArH), 8.26 (d,
2H, J = 11.2 Hz), 8.47 (s, 1H, CH=N−), 10.39 (s, 1H, −NH).13C NMR (DMSOꢀd6, 100 MHz) δ
14.7, 63.4, 106.0, 112.6, 113.3, 115.5, 116.0, 118.0, 123.5, 124.7, 125.4,126.2, 126.2, 126.6,
127.4, 128.4, 130.8, 131.8, 133.4, 146.6, 148.9, 153.4, 160.1, 162.3 (CH=N−), 164.8, 181.3.
HRMS (m/z): 552.2543. Calc. for C29H21ClN6O2S (%): C, 62.98; H, 3.83; N, 15.20; Found: C,
62.96; H, 3.81; N, 15.22.

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2.5.9. General procedure for Synthesis of amino thiazole and morpholino based s-triazine
Schiff base derivatives (13a-c).
Ionic liquid BMIM [Tf2N] (l equiv), (1 equiv) was added to a solution of (E)ꢀNꢀ(4ꢀ
chloroꢀ6ꢀ(4ꢀ((6ꢀethoxybenzo[d]thiazolꢀ2ꢀylimino)methyl)phenoxy)ꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ6
ethoxybenzo[d]thiazolꢀ2ꢀamine (12a) (1 equiv) and morpholine 2 (1 equiv). The reaction mixture
was taken in a two necked round bottomed flask fitted with reflux condenser. To this, 1 equiv
KOH and dry Acetone/ DMF (2:1, 20 mL) were added and the reaction mixture was refluxed at
0
50 C for 1ꢀ2 h. The progress of the reaction was monitored by TLC. Upon completion of the
reaction, the mixture was allowed to cool to room temperature. The resulting residue washed
with THF and water (1:5) to afford the pure compounds (13aꢀc).
(E)-6-ethoxy-N-(4-((4-((6-ethoxybenzo[d]thiazol-2-yl)amino)-6-morpholino-1,3,5-triazin-2-
yl)oxy)benzylidene)benzo[d]thiazol-2-amine (13a).
Green solid, yield: 96%. Mp: >300 ºC; IR (KBr, cmꢀ1) 3200, 2964, 2915, 1577, 1361.1H
NMR (DMSOꢀd6, 400 MHz) δ: 1.34 (s, 6H, −CH2−CH3), 3.64 (s, 4H, −O−CH2), 3.64 (s, 4H,
−N−CH2), 3.98(−CH2−O− merged with DMSO solvent peak), 6.80 (d, 2H, J = 8.0 Hz, ArH),
6.95 (d, 2H, J = 7.6 Hz, ArH), 7.17 (s, 2H, ArH), 7.26 (d, 2H, J = 8.0 Hz, ArH), 7.76 (d, 1H, J =
7.2 Hz), 8.13 (s, 1H, ArH), 9.77 (s, 1H, −N=CH), 10.65 (s, 1H, −NH). 13C NMR (DMSOꢀd6, 100
MHz) δ; 14.5, 43.6, 63.5, 65.7, 105.8, 113.3, 115.8, 117.9, 124.1, 126.5, 128.0, 131.5, 132.0,
146.0, 150.7, 153.5, 163.5, 165.1, 177.2, 191.0. HRMS (m/z): 654. 7500. Calc. for
C32H30N8O4S2 (%): C, 58.70; H, 4.62; N, 17.11; Found: C, 58.72; H, 4.51; N, 17.20.

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(E)-6-ethoxy-N-(4-morpholino-6-(4-((phenylimino)methyl)phenoxy)-1,3,5-triazin-2-yl)
benzo[d]thiazol-2-amine (13b).
Light yellowish green solid, yield: 95%. Mp: >250 ºC; IR (KBr, cmꢀ1) 3211, 2968, 2922,
1580, 1361. 1H NMR (DMSOꢀd6, 400 MHz) δ: 1.36 (s, 3H, −CH2−CH3), 3.67 (t, 4H, −O−CH2),
4.01 (s, 4H, −N−CH2), 4.02 (s, 2H, ꢀOꢀCH2), 6.94 (d, 2H, J = 6.8 Hz, ArH), 6.94 (s, 1H, J = 6.8
Hz, ArH), 7.30 (d, 2H, ArH), 7.35 (t, 1H, ArH), 7.35 (s, 1H, ArH), 7.41 (d, 2H, ArH), 7.41 (d,
1H, ArH), 7.72 (d, 2H, J = 7.2 Hz), 9.77 (s, 1H, −CH=N−) 10.65 (s, 1H, −NH), 13C NMR
(DMSOꢀd6, 100 MHz) δ; 14.4, 43.3, 63.6, 65.5, 107.1, 114.8, 115.6, 116.1, 120.6, 122.0, 126.5,
1272, 128.1, 129.1, 129.3, 131.7, 149.7, 151.6, 155.3, 163.3, 163.7, 168.7, 190.3. Calc. for
C29H27N7O3S (%): C, 62.91; H, 4.92; N, 17.71; Found: C, 62.87; H, 4.95; N, 17.72.
(E)-6-ethoxy-N-(4-morpholino-6-(4-((naphthalen-1-ylimino)methyl)phenoxy)-1,3,5-triazin-
2-yl)benzo[d]thiazol-2-amine(13c).
Light solid, yield: 95%. Mp: 282ꢀ284 ºC IR (KBr, cmꢀ1) 3172, 3055, 2967, 1601, 1582,
1
1367. H NMR (DMSOꢀd6, 400 MHz) δ: 1.39 (s, 3H, ꢀCH2ꢀCH3), 3.59 (t, 4H, ꢀOꢀCH2), 3.72 (s,
2H, ꢀOꢀCH2), 4.99 (t, J = 6.0 Hz, 4H, ꢀNꢀCH2), 6.94 (d, 2H, J = 7.6 Hz, ArH), 6.94 (d, 1H, J =
7.6 Hz, ArH), 7.41 (d, 2H, J = 8.0 Hz, ArH), 7.50 (s, 1H, ArH), 7.60 (s, 1H, ArH), 7.60 (s, 1H,
ArH), 7.75 (d, 2H, J = 7.6 Hz, ArH), 7.78 (s, 1H ArH), 7.93 (d, 1H, J = 7.6 Hz, ArH), 7.96 (d,
13
1H, J = 7.2 Hz), 8.27 (d, 1H, J = 6.0 Hz), 9.60 (s, 1H, ꢀNH), 9.77 (s, 1H, CH=Nꢀ). C NMR
(DMSOꢀd6, 100 MHz) δ 14.5, 43.4, 44.2, 63.7, 65.4, 65.6, 107.7, 115.3, 115.1, 115.8, 117.9,
122.0, 123.1, 123.4, 125.7, 126.3, 126.7, 127.6, 128.2, 128.3, 131.2, 132.0, 133.4, 133.7, 149.9,
155.3, 163.3, 163.7, 168.7, 169.1, 190.9. Calc. for C33H29N7O3S (%): C, 65.65; H, 4.84; N,
16.24; Found: C, 65.60; H, 4.89; N, 16.19.

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3. Result and Discussion
3.1. Synthesis and spectral characterization of s-Triazinyl Schiff base derivatives
The cyanuric chloride (CC) and its derivatives (i.e., mono, bis, trichloride) are known to
be reactive very easily with various nucleophiles such as amines, alcohols, and thiols (Fig. 1).19,
45ꢀ56
As an initial attempt to find an optimal reaction condition, a variety of experimental
conditions were examined for cyanuric chloride (1) by changing catalyst, temperature, and
reaction medium. The synthesis of various 1,3,5ꢀtriazineꢀbased mono and bisꢀmorpholine amino
derivatives and were carried out, starting from 2,4,6ꢀtrichloroꢀ1,3,5ꢀtriazine and ends with a
nucleophile. Trisubstituted 1,3,5ꢀtriazine derivatives can be generated by stepwise replacement
of the chlorine atoms of CC with amino thiazole/morpholine (–NH) and Schiff bases (–OH). The
synthetic pathway of mono (5a-d and 8a-c) and diꢀmorpholine based sꢀtriazine Schiff’s bases
(6a-c and 9a-b), amino thiazole based sꢀtriazine Schiff’s bases (12a-c), and their derivatives
(13a-c) are illustrated in Scheme 1 and 2. Synthesis of starting materials, namely monoꢀ
substituted morpholino sꢀtriazine derivative (3) and Schiff’s bases (4a-d and 7a-c) were
accomplished based on the reported literature.45, 46 All the compounds were purified by column
1
chromatography and the products were characterized by IR, H, 13C NMR, Mass spectra and
elemental analyses and the observed values were consistent with the assigned structures. 2D
NMR spectra were recorded for compounds 6a (Figs. S5-7) and 12b (Figs. S11-15)
(Supplementary data (S)) and the remaining compounds and their signals were assigned based on
the spectral assignments made by the compounds 6a and 12b. Resulting molecular ion peaks and
the elemental analysis results are in good agreement with the molecular formula of the target

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molecules. Every molecule gives a molecular ion peak [M+ 1] with low intensity and a base peak
near the molecular weight of the Schiff bases used.
3.1.2. IR and NMR Spectral characterization of newly synthesized compounds
The structure of newly synthesized compounds was supported by spectral analyses such
as FT–IR, HR–Mass, 1D and 2D NMR spectra. The bisꢀmorpholino based sꢀtriazine Schiff base
derivative of compound 6a shows characteristic band in the IR region at 1576 cmꢀ1assignable to
−C=N– stretching vibration of sꢀtriazine ring and the –CT–O– (TCꢀTriazine carbon) absorption is
observed as a distinct band at 1361cmꢀ1(6a).57, 58 Besides, the −NH−TC− broad band observed at
3216 cmꢀ1 in IR spectrum, reveals that the presences of thiazole moiety in sꢀtriazine scaffold of
compound 12b. Furthermore, sꢀtriazine derivatives show another important band in the region
1577–1586 cmꢀ1 is ascribed to −C=N stretching vibrations of sꢀtriazine ring.59 Hence, the IR
spectral data illustrate the formation of the 1,3,5ꢀtriazine Schiff base derivatives.
Compounds 6a and 12b (Fig. 2) were taken as a representative compound and the signals
were assigned based on the correlations in the 2D spectra. The 1D (1H &13C NMR) and 2D NMR
(HMBC) spectra of compound 6a are shown in Figs. S5-7 for the remaining compounds, the
signals were assigned based on the spectral values compound 6a.
1
In H NMR spectrum of 6a, the singlet at 9.76 ppm is assignable for azomethine (–
CH=N) proton. Besides the absence of –OH and –NH peaks confirm the condensation between
Schiff base and morpholine to form sꢀtriazine scaffold derivatives. In 13C spectra, peaks at 191.0
ppm and 163.7 ppm could be unambiguously assigned to Cꢀ6 (−TC−O−) and Cꢀ2/Cꢀ4 (−TC−N)
of 2,4,6ꢀtriazine carbons, and the azomethine (–CH=N) carbon absorbs at 163.4 ppm (Cꢀ14).

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All the other aliphatic and aromatic protons and carbon of 6a were observed in the
expected regions. Further, this assignment was substantiated by HMBC analysis. The HMBC
spectrum reveals that the signal at 7.75 ppm (Cꢀ9 hydrogen) showing γ correlation with Cꢀ6
sꢀtriazine carbon at 191.0 ppm, and azomethine carbon (–CH═N) at 163.4 ppm (Fig. S8). In
addition, The Cꢀ2'' and Cꢀ4'' protons also show β correlation with Cꢀ2 and Cꢀ4 sꢀtriazine carbons
at 163.7 ppm. Based on the heteronuclear correlation, compound 6a confirms the formation
morpholine based sꢀtriazine Schiff base scaffold derivatives.
1
1
Moreover, in H and Hꢀ1H COSY NMR spectra of 12b (Figs. S11 & S13), one broad
singlet at 10.78 ppm with one proton integral and one sharp singlet at 9.76 ppm with one proton
integral, were assigned to secondary amine (−NH) proton and (–CH=N) azomethine proton,
respectively. Further, this assignment was substantiated by HSQC analysis (Fig. S14). In the 13C
NMR spectrum of compound 12b (Fig. S12), the signal at 163.3 ppm is assigned for (–CH=N)
azomethine carbon of the Schiff base moiety. In addition, signals at 190.9, 154.3 and 149.8 ppm
were unambiguously assigned to Cꢀ2 (−TC−NH−), Cꢀ4 (−TC−O−) and Cꢀ6 (−TC−Cl) of 2,4,6ꢀ
triazine carbons. The other chemical shift values observed at 115.83, 120.8, 126.4, 129.5, 132.0,
and 163.3 ppm are assigned for (Cꢀ9, Cꢀ19, Cꢀ17, Cꢀ18, Cꢀ10 and Cꢀ14 (–CH═N) carbons of the
Schiff base moiety) shows one bond correlation with signal at 6.96, 7.40, 7.27, 7.40, 7.74, 9.76
ppm. In addition, the HMBC (Fig. 15) spectrum reveals that the signal at 7.74 ppm (132.0 ppm,
Hꢀ9 hydrogen) shows gamma correlation with Cꢀ6 and Cꢀ14 at, sꢀtriazine carbon at 190.9 ppm
and 163.3 ppm (−C═NH) azomethine carbon. All other aliphatic and aromatic protons and
carbons of 12b gave their characteristic signals at the expected regions as illustrated in the
Figs. S11- 15. The 2D NMR (1Hꢀ1H COSY, HSQC, and HMBC) analysis of 12b confirmed its
structure of without ambiguity.

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3.2. Computational Results
All the synthesized compounds were screened by using Qikprop to find the ADME
properties and the results were tabulated in Table 3. The Lipinski’s rule of five values of
compounds 5a-13c indicates that all the compounds are endowed with a drug like properties
except compound 12a-c and 13a-c. Compounds 5a, 5b, 5d, 6b, 8a-c, and 9a showed no violation
of Lipinski’s rule of five. However, compounds 5c, 6c & 9b showed one violation, compounds
12a-c & 13b-c showed 2 violations and compound 13a showed 3 violations and their partition
coefficient value between octanol and water is more than 5 and the molecular weight is greater
than 500 amu. All the tested compounds have satisfactory percentage (82.215ꢀ 100%) of human
oral absorption and predicted aqueous solubility (QPlogS) parameter indicated that the
compounds 5a-d, 6a-c, 8a-c, 9a, and 9b possess permissible values and the 6ꢀ
ethoxybenzo[d]thiazolꢀ2ꢀamine substituted compounds (12a-c, and 13a-c) hold low (poor)
aqueous solubility. Likewise, the predicted bloodꢀbrain barrier permeability (QPlogBB) for the
tested compounds was assessed and exhibit satisfactory values. The IC50 value of HERG K+
channel blockage (QPlogHERG) was also predicted and the results showed that all the tested
compounds have an admirable range of values.
Docking studies were carried out in order to evaluate the binding affinity and the
interactions between the synthesized compounds and the P53 protein (PDB ID: 4OGN). The
interactions between 4OGN and the compounds are tabulated in Table 4 and all the figures are
given supplementary data. From the docking simulation analysis, the docking score of the
compounds 5a-13c varies from ꢀ9.148 to ꢀ4.388. All the synthesized sꢀtriazine scaffolds have
good glide score with significant interaction with the essential residue of HIE 96 and HIE 73 and

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compounds 5a (GLN 59) and 5c (GLN 59, LYS 70). Moreover, compounds 5d, 6a, 6c, 8c, 9a, 12a,
12c, and 13a exhibit good π−π interaction with crucial residues of HIE 96 by which is essential
for the inhibitory affinity of p53.60 These π−π stacking interactions were performed by the 1,3,5ꢀ
triazine ring (5d, 6a, 6c, 8c, and 9a) and 6-ethoxybenzo[d]thiazolꢀ2ꢀamine moiety (12a, 12c, and
13a). Preliminary SAR (Structure−activity relationship) studies suggested that the presence of
1,3,5ꢀtriazine moiety is essential for the interaction between the p53 and compounds, and that of
a smaller group on the ethoxybenzo[d]thiazolꢀ2ꢀamine shows beneficial for the binding efficacy.
Moreover, the structural investigation highlighted that the presence of 3ꢀfluoro substituted Schiff
moiety in the sꢀtriazines confirms the increase in binding efficacy (ꢀ7.741 to ꢀ9.148). Structural
analysis of endogenous sꢀtriazines, the presence of naphthalene and phenyl groups of Schiff base
moiety and morpholine ring, substituted at the 1,3ꢀ5ꢀtriazine ring are necessary for the activity as
hydrophobic and amphiphilic regions, respectively.
The docking result clearly indicates the water molecules present in the active site of p53
protein plays an important role in binding. All the sꢀtriazine derivatives except 5c, 8c, 12b, and
13b showing the interactions with one or two water molecules present in the active site. The
Vander Waals energy (evdw) and Coulomb energy (ecoul) are the predicted Vander Waals
interaction and electrostatic interaction energies between a ligand and the protein. The
interactions between the p53 protein and the compounds 5c and 6c are shown in Fig. 3.
3.3. Antitumor Studies
3.3.1. In vitro assay for evaluation of cytotoxic activity
The cytotoxicity of the tested compounds (5a-d, 6a-c, 8a-c, 9a-b, 12a-c, and 13a-c) were
evaluated against human fibro sarcoma tumor cell line (HTꢀ1080) and cervical cancer (HeLa

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cell) at various concentrations viz. 5, 10, 20, 30, 40, 50 and 60 ꢁg/mL with 5ꢀfluorouracil taken
as standard drug are tabulated in Table 5. Figs. 4 and 5 shows the inhibitory effect of the
synthesized compounds 5a-d, 6a-c, 8a-c, 9a-b, 12a-c, and 13a-c on HTꢀ1080 cell and HeLa
tumor cells after 24 h and 48 h, respectively. Treated cells have shown shrinkage in volume of
cells, membrane blebbing and loss of cell adhesion. The screening results have revealed that the
tested compounds exhibited good and moderate antitumor activity against both cell lines
compared to the standard drug.
Among this series, compounds 5c, 5d, 6c, 12c, and 13c have shown superior activity
against fibro sarcoma tumor cells (HTꢀ1080) compared to reference drug with IC50 values 19±
0.76, 20± 0.60, 18± 0.82, 23± 0.30 and 23± 0.81 ꢁg/mL for 24 h and 11± 0.52, 12± 0.32, 10±
0.45, 12± 0.57 and 10± 0.61ꢁg/mL for 48 h, respectively. It was observed that the naphthalene
ring containing sꢀtriazine compounds (5c, 6c, 12c, and 13c) plays an important role in enhancing
the cytotoxicity of these compounds.
The compounds 5a, 5b, 6a, 6b, 8a, 8c, and 12a with benzothiazole based sꢀtriazine
pattern have displayed moderate anticancer activity, whereas the remaining compounds have
shown lower anticancer potency when compared to standard drug 5ꢀfluorouracil. The structural
activity profile shows that the naphthalene moiety substitution in the sꢀtriazine ring is important
to enhance the anticancer activity. Besides, bisꢀmorpholine substituted sꢀtriazine compound 6c
(IC50=10± 0.45ꢁg/mL) was found to be more potent when compared with compound 5c
(IC50=11± 0.52 ꢁg/m) against HTꢀ1080 cell line. It has been observed that an enhanced antitumor
activity was established by additional morpholine moiety in the sꢀtriazine ring as an alternative
of two chlorine atom.

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Besides, the introduction of 3ꢀfluoro based Schiff moiety in the 1,3,5ꢀtriazine Cꢀ4
position (Compounds 8a-c and 9a-b) indicates slightly decreasing the anticancer activity except
compound 8a. However, the replacement of the morpholine group in Cꢀ2 position at sꢀtriazines
of hit 5c (IC50=19± 0.76 ꢁg/mL) by a 6ꢀethoxybenzo[d]thiazolꢀ2ꢀamine of compound 12c (IC50
=23± 0.30 ꢁg/mL) led to the decreased activity. The results revealed that the chlorine atom is not
influenced any cytotoxic activity and the structural activity relationship (SAR) analysis
demonstrated that the type of the substitutions are significant for the inhibitory activity.
Also the in vitro antitumor evaluation of the synthesized compounds 5a-d, 6a-c, 8a-c, 9a-
b, 12a-c, and 13a-c were screened for in vitro anticancer activity of HeLa tumor cells with
5ꢀfluorouracil standard drug. Compounds 5d and 6a have shown more potent against HTꢀ1080
cells compared to reference drug with IC50 values 23± 0.50 and 26± 0.70 ꢁg/mL for 24 h and 10±
0.62 and 11± 0.40 ꢁg/mL for 48 h, respectively. Moreover, compounds 8c and 13a have shown
similar activity than reference drug, whereas the rest of the compounds have demonstrated lesser
potency or did not show any activity (9a) even at maximum concentration.
3.2.2. Evaluation of reactive oxygen species (ROS) of Compounds 5c, 5d and 6c.
Levels of ROS in control and compounds of 5c, 5d, and 6c cells were depicted in Fig. 6.
sꢀtriazine derivatives (5c, 5d, and 6c) treatment caused a rapid increase of intracellular ROS in
HTꢀ1080 cells. A significant increase in the ROS levels in50 ꢁg/mL of sꢀtriazine derivatives
treated cells was further noticed. The increased ROS levels during the sꢀtriazine compounds
treatment might be due to its proꢀoxidant property. Production of ROS leads to an increase in
green fluorescence intensity correlated with an increase in apoptosis. DCFH–DA (2′,7′ꢀ
dichlorodihydrofluorescein diacetate) staining (Fig. 6) shows that DCFH–DA is cleaved in an

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intracellular medium by nonꢀspecific enzymes to form DCF (dichlorofluorescein) which is
further oxidized by ROS to form the green fluorescent compound. The change in the
fluorescence intensities reflects a measure of change in the relative levels of intracellular ROS.
3.2.2. Evaluation of mitochondrial membrane potential (Rhodamine-123).
The authors have further observed changes in the mitochondrial membrane potential in
compounds 5c, 5d, and 6c treated and control cells (Fig. 7). A mitochondrion is very important
organelles in regulating the cells of death as well as a maker of apoptosis.61 The authors have
observed that the mitochondrial membrane potential has been altered during 5c treatment. This
shows that the mitochondrial membrane potential plays a role in 5c induced cell death.62 The
authors have observed that 5c pretreatment significantly increased apoptotic morphological
changes in HTꢀ1080 cells. Mitochondrial membrane potential (ꢂΨm) is probed by using Rhꢀ123
(Rhodamineꢀ123), a lipophilic cationic dye. Mitochondria are involved in the regulation of
chemotherapyꢀinduced apoptosis. Loss of ꢂΨm leads to morphological changes. Rhꢀ123
distributes itself according to the negative potential across the mitochondrial inner membrane
and gives a green fluorescence with respiring (living) mitochondria (Fig. 7). With an increase in
dosage, there is a decrease in ꢂΨm. This resulted in a loss of the dye Rhꢀ123, indicated by a
decrease in fluorescence intensity, and has shown that mitochondrial ROS play a vital role in
apoptosis. Similar results were obtained with 5d and 6c.
3.2.3. Evaluation of Apoptosis (AO/EtBr Staining)
Apoptotic morphological changes were followed by acridine orangeꢀethidium bromide
dual staining (AO/EtBr Staining) method. HTꢀ1080 cells treated with an IC50 dose of the
synthesized compounds (5c, 5d, and 6c) revealed apoptotic characteristics such as loss of

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cytoplasmic membrane integrity thereby uptake of ethidium bromide making the nucleus stain
red. Ethidium bromide dominates AO in apoptotic cells. The loss of cytoplasmic membrane
integrity is used as an indicator of cell viability. Whereas, the untreated HTꢀ1080 cells were
stained green as a result of intercalation into the doubleꢀstranded DNA. From the histograms and
fluorescent microscopic pictures (Fig. 8), it is clear that 5c induced apoptosis pathway, even
prescribed amount concentration of compound 5a. In control, live cells with normal green nuclei
were seen.
Early apoptotic cells have shown bright green nucleus with condensed or fragmented
chromatin and late apoptotic cells have displayed condensed and fragmented orange or red
colored chromatin. Cells that have died from direct necrosis have a structurally normal orange
nucleus. Analysis of apoptotic characters by AO/EtBr staining in HTꢀ1080 cell line treated with
newly synthesized compounds (5c, 5d, and 6c) using fluorescence microscopy is shown in
Fig. 8. The treatment of compounds 5c, 5d and 6c has caused more cells to undergo death in HTꢀ
1080 liver cancer cells, in agreement with the cytotoxic results. The observations have revealed
that all the compounds treated cells show cytological changes, whereas, in the untreated cells, no
such changes were observed. The photomicrographs in Fig. 8 show viable (light green), early
apoptotic (bright green), late apoptosis (red to orange) and necrosis (red) in 5c, 5d, and 6c treated
cancer cells.
4. Conclusion
In summary, a convenient and efficient method to synthesize sꢀtriazine derivatives with
good yields has been reported. The synthesized compounds were characterized by elemental
analysis, FT– IR, HR–Mass and 1D/2D NMR. All the spectral data unambiguously demonstrates
the formation of mono, bis, and triꢀsubstituted 1,3,5ꢀtriazine derivatives. The various substitution