Synthesis and in vitro evaluation of novel triazine analogues as anticancer agents and their interaction studies with bovine serum albumin

Article abstract of DOI:10.1016/j.ejmech.2016.03.088

A novel series of triazine-benzimidazole analogs has been designed and synthesized for their in vitro anticancer activities. Four compounds (6, 16, 17 and 20) were identified as highly potent anticancer agents against 60 human cancer cell lines with GI₅₀ in the nanomolar range. To improve the drug applications toward cancer cells, there is a need to couple these compounds to some carrier macromolecules. Following this approach, the interaction between triazine-benzimidazole analogues and bovine serum albumin (BSA) has been investigated with UV-Visible and fluorescence spectroscopic methods under physiological conditions. The observed fluorescence quenching indicates that these compounds could efficiently bind with BSA and be transported to the target site.

Full text of DOI:10.1016/j.ejmech.2016.03.088

Accepted Manuscript
Synthesis and in vitro evaluation of novel triazine analogues as anticancer agents and
their interaction studies with bovine serum albumin
Prinka Singla, Vijay Luxami, Kamaldeep Paul
PII:
S0223-5234(16)30271-9
10.1016/j.ejmech.2016.03.088
EJMECH 8513
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2016
Revised Date: 30 March 2016
Accepted Date: 31 March 2016
Please cite this article as: P. Singla, V. Luxami, K. Paul, Synthesis and in vitro evaluation of novel
triazine analogues as anticancer agents and their interaction studies with bovine serum albumin,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.088.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and in vitro evaluation of novel triazine analogues as anticancer
agents and their interaction studies with bovine serum albumin
Prinka Singla, Vijay Luxami* and Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar University, Patiala-147 004, INDIA

ACCEPTED MANUSCRIPT
Synthesis and in vitro evaluation of novel triazine analogues as anticancer
agents and their interaction studies with bovine serum albumin
Prinka Singla, Vijay Luxami* and Kamaldeep Paul*
School of Chemistry and Biochemistry, Thapar University, Patiala-147 004, India
*E-mail: kpaul@thapar.edu (K. Paul), vluxami@thapar.edu (V. Luxami)
Abstract
A novel series of triazine-benzimidazole analogs has been designed and synthesized for their
in vitro anticancer activities. Four compounds (6, 16, 17 and 20) were identified as highly
potent anticancer agents against 60 human cancer cell lines with GI50 in the nanomolar range.
To improve the drug applications toward cancer cells, there is a need to couple these
compounds to some carrier macromolecules. Following this approach, the interaction
between triazine-benzimidazole analogues and bovine serum albumin (BSA) has been
investigated with UV-Visible and fluorescence spectroscopic methods under physiological
conditions. The observed fluorescence quenching indicates that these compounds could
efficiently bind with BSA and be transported to the target site.
Keywords: Triazine, Benzimidazole, Antitumor activity, Bovine serum albumin,
Fluorescence resonance energy transfer.
1. Introduction
Cancer is considered one of the world’s most devastating disease [1]. Millions of new cases
are reported every year with high mortality. Improvements in treatment and prevention led to
diminish the cancer deaths. Considerable efforts are given to identify molecules with
anticancer properties from both natural and synthetic sources [2]. It is well-known that
heterocyclic compounds are abundant and their structural subunits exist in many natural
products such as vitamins, hormones, antibiotics, alkaloids and pharmaceuticals. Among the
wide variety of synthetic compounds recognized as potential anticancer drugs, molecules
based on triazine [3] and benzimidazole [4] scaffolds have attracted great interest. Several
reports describing evaluation of triazine derivatives as antitumor agents appeared in the
recent literature [5-7]. For example, HL010183 [8] and irsogladine [9], possess
antiangiogenic and anticancer properties. Similarly, lysophosphatidic acid acyltransferase-β
inhibitor (CT32228), demonstrates a very good antileukemic profile [10]. The triazine ring in

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altretamine (hexamethylmelamine) is typically responsible for anticancer activity [11].
Gedatolisib with triazine ring has been found to be the dual phosphoinositide 3-kinase and
mTOR kinase inhibitors, which is currently under clinical trials [12]. This structural motif is
also present in reversing anticancer multidrug transport inhibitor of ABCG2 transporter (PZ-
39) [13] and in an aromatase inhibitor (SEF19) (Figure 1) [14]. Herein, our strategy is to
discover new cancer chemotherapeutic agents using a small molecule library that contained
triazine and benzimidazole core structures.
Figure 1
It has been observed that low antiproliferative activity of heterocyclic compounds is due to
restricted access to the target site. This limitation has hindered the potential clinical use of
heterocyclic compounds. A promising approach to improve their efficacy towards cancer
cells is to couple anticancer drugs to carrier macromolecules. Various types of
macromolecules have been used including poly(ethyleneglycol) polymer, nanoparticles,
nanotubes, liposomes, dendrimers and protein biomolecules [15]. Among these, bovine serum
albumin (BSA) is used as a promising drug delivery system owing to abundance in plasma.
Serum albumin transporter acts as carriers of endogenous and exogenous compounds [16]
including fatty acids, amino acids, drugs, and pharmaceuticals for improvement of
pharmacokinetic and pharmacodynamic behavior of several drugs. In addition, owing to its
biochemical and pharmacological properties, BSA has also been used to target diseased and
malignant cells as a versatile drug carrier; resulting in higher efficacy of treatment and
reduced side effects [17-19]. The binding pocket of BSA, with two tryptophan residues (Trp-
134 and Trp-212) and phenylalanine possesses intrinsic fluorescence [20] and binding
changes the fluorescent signal, providing a convenient method to understand structural
changes. Therefore, in the present study we have studied the interactions of triazine-
benzimidazoles and BSA by using UV-Visible and fluorescence spectroscopic techniques.
This method further allowed the determination of binding parameters such as binding
constant, number of binding sites, and binding distance of compounds and BSA, which
helped in elucidate the binding probability.
2. Results and Discussion
2.1. Chemistry
Triazine-benzimidazoles (5-21) were prepared as shown in scheme 1. Cyanuric chloride was
o
stirred with morpholine in the presence of sodium bicarbonate in THF at 0-5 C for 6 h to
give 4,6-dichloro-2-morpholin-4-yl-[1,3,5]triazine (2) in 89% yield. Condensation of 4-

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aminobenzoic acid with o-phenylenediamine in the presence of polyphosphoric acid at 200
oC for 5 h afforded 4-(1H-benzimidazol-2-yl)-phenylamine (3) as white solid. Compound 2
was underwent nucleophilic substitution reaction with 4-(1H-benzimidazol-2-yl)-
phenylamine (3) in the presence of potassium carbonate and THF at room temperature for 24
h, furnished [4-(3H-benzimidazol-5-yl)-phenyl]-(6-chloro-2-morpholin-4-yl-[1,3,5]triazin-2-
yl)-amine (4) in 84 % yield. Further, treatment of compound 4 with different primary and
secondary amines in the presence of potassium carbonate and 1,4-dioxane at 110 oC for 8-12
h gave compounds 5-15 in 64-78% yields. Various five and six member aryl groups like
furan, thienyl and phenyl were also substituted with compound 4 via Suzuki-Miyuara cross
coupling reaction in the presence of 10 mol% of Pd(PPh3)4 and 1.5 equivalents of K2CO3 in
1,4-dioxane to afford compounds 16-21 in good yields (65-72%). The structures of newly
1
13
synthesized compounds were confirmed by H and C NMR as well as mass spectrometry
(Supporting Information).
Scheme 1
2.2.
In Vitro anticancer screening
Compounds 5-21 were submitted to the National Cancer Institute (NCI), Bethesda, Maryland,
USA for evaluation of antitumor activities. Four compounds (6, 16, 17 and 20) were selected
for in vitro studies against 60 human cancer cell lines at one dose concentration of 10 µM.
Structures are generally selected for screening based upon drug-like properties utilizing the
concept of privileged scaffolds or structures based on computer-aided design. In vitro
antitumor screening data revealed that triazine with piperidine (6), phenyl (16), 4-
fluorophenyl (17) and 4-chlorophenyl (20) substitutions showed significant inhibitory activity
towards most of the cancer cell lines. Compounds 6, 16, 17 and 20 were found to be broad
spectrum against all nine subpanels of cancer cell lines at primary single dose concentration
(10 µM) with mean growth percent of 0.72, -56.41, -7.32 and -83.19 respectively. Close
examination of the data presented in Table 1 indicated that compounds 16 and 20 have been
found to be most potent while 6 and 17 showed moderate antitumor activities.
Table 1
These four compounds were further screened against cell lines representative of the 9
different cancer types, at five doses (0.01-100 µM) to determine GI50, TGI and LC50. Results
indicated these compounds were very effective against two leukemia cancer cell lines: HL-60
(TB) and SR, with GI50 values in the nanomolar range. HL-60 (TB) appeared to be the most
sensitive to compound 16, exhibiting a GI50 value of 31 nM with TGI value of 601 nM.

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Similarly, compounds 6, 16, 17 and 20 also showed effective growth inhibition against
leukemia cancer cell line SR with GI50 values of 731 nM, 125 nM, 539 nM and 31 nM,
respectively. All four compounds were also active against renal cancer cell line RXF393 with
respective GI50 values of 808 nM, 501 nM, 459 nM and 222 nM. Compound 20 (MG MID
GI50 value of 720 nM) is almost four fold, two fold and three fold more active than
compounds 6, 16 and 17 with MG MID GI50 values of 2680 nM, 1380 nM and 2370 nM
respectively (Table 2 and Figures S39-S54) [21-24].
Table 2
2.3.
Bovine serum albumin interactions.
To examine the structural interaction of bovine serum albumin (BSA) with compounds 5-21,
absorbance and emission spectral measurements were carried out. The UV-visible spectrum
of BSA (10 ꢀM) showed an intense band at 279 nm in phosphate buffer at pH 7.4 that arises
due to the presence of phenyl rings in Trp (tryptophan), Tyr (tyrosine), and Phe
(phenylalanine) residues. Subsequent addition of compounds 5-14, 16, 17 and 20 to BSA
solution resulted in a gradual increase in the intensity of peak at 279 nm with the appearance
of new band at 324 nm. These changes in absorbance clearly indicated the interaction of
compounds with BSA and leading to an increase of hydrophobicity in the vicinity of Trp, Tyr
and Phe residues (Figure 2). As shown in Table 3, the addition of compounds 5-14, 16, 17
and 20 to BSA resulting in hyperchromicity within range of 65.29% - 97.67% (Figures S55-
S63), while compounds 15, 18, 19 and 21 did not show any significant change with BSA.
Moreover, the maximum absorption wavelength of BSA remains unchanged, implying that
the interactions between compounds and BSA are non-covalent in nature and likely occur
through π−π stacking between aromatic rings of compounds and phenyl rings of Trp, Tyr and
Phe residues located in the binding cavity of BSA.
Figure 2
These interaction studies were further investigated through fluorescence spectroscopy. On
excitation at 280 nm, the emission spectrum showed an intense band at 350 nm. The emission
is sensitive to changes in local environment of the Trp-134 and Trp-212 and so can be
attenuated by binding of a small molecule at or near this residue. In all cases, 350 nm
fluorescence emission of the protein was decreased with increasing concentrations of
compounds 5-14, 16, 17 and 20, indicating the binding of compounds to the protein. The
decrease in the fluorescence intensity of BSA at 350 nm was accompanied by the

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enhancement of new band within the range of 370 nm and 373 nm, which is attributed to
formation of a complex between compounds and protein (Figure 3 and Figures S55-S63).
Figure 3
These absorption and emission results indicated that the microenvironment of the three
aromatic acid residues might be altered and the tertiary structure of BSA destroyed.
Compounds 15, 18, 19 and 21 did not show any significant change with the conformation of
BSA, indicating for the requirement of linker or heteroatom at C-6 position of triazine. As a
result all further testing was carried out on compounds 5-14, 16, 17 and 20. The changes in
absorption and fluorescence intensities clearly indicated the interacting properties of triazine-
benzimidazoles with BSA. When a small molecule binds independently to a set of equivalent
sites on a macromolecule, the photophysical properties, binding constants (determined by
Benesi–Hildebrand equation) [25] and the numbers of binding sites (n) (using double-
logarithmic equation) [26,27] can be determined. In this way, we quantitated the binding
strength between triazine-benzimidazole analogues and BSA. The binding constant of
complexes confirmed a significant role for the configuration of triazine-benzimidazole
analogues around BSA as shown in Table 3. The linear fitting plots of respective 1/(A0-A)
and 1/(F0-F) vs. 1/[compounds] (Inset Figures 2 and 3) were observed for the interaction
between BSA and compounds 5-14, 16, 17 and 20 (Figures S55-S63). The value of n is
helpful to know the number of binding sites for compounds and BSA which in most of the
cases has been found to be nearly 1. Thus, there was one independent class of binding site on
BSA for compounds, and the molar ratio of protein to compounds 5-14, 16, 17 and 20 is 1:1
in the binding reactions.
Table 3
2.4.
Energy transfer between triazine-benzimidazole analogues and BSA
To further confirm the vicinity of the analogues to the BSA (distance between compound and
BSA) [28,29], fluorescence resonance energy transfer (FRET) from protein to ligands was
verified. According to the Förster theory of non-radioactive energy transfer, the transfer of
energy can take place through a direct electrodynamic interaction between the primarily
excited molecule and its neighbour. Using the fluorescence resonance energy transfer
(FRET), the distance r of binding between compounds 5-14, 16, 17 and 20 and BSA has been
calculated by the equation-1 [30].

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R06
F
E =1−
=
--------1
F0 R06 + r6
where E denotes the efficiency of energy transfer between donor and acceptor, r is the
distance between donor and acceptor, and F and F0 are fluorescence intensities of BSA in the
presence and absence of quencher, respectively. R0, the critical energy transfer distance when
the transfer efficiency is 50%, is given by the equation-2 [31].
R0 = 8.79x10−25
[
K 2n−4ΦJ
]
---------2
K2 is the orientation factor of the donor and acceptor dipoles, n is the refraction index of
medium, Φ is the fluorescence quantum yield of the donor in the presence of acceptor, and J
expresses the degree of spectral overlap between the donor emission and the acceptor
absorption, which could be calculated by the equation-3:
∞ F(
λ
)
ε
(
λ
)
λ
λ
4d
λ
∫
0
J =
-----3
∞ F(
λ
)d
∫
0
where F(λ) is the normalized donor emission spectrum in the range from λ to λ + ꢀλ, and ε(λ)
is the molar absorption coefficient of the acceptor at wavelength λ. According to the above
equations (eq. 1-3) and using K2 = 2/3, n = 1.33 and Φ = 0.15 [32], J, R0, r, and E were
calculated (Table 4). On gradual addition of compounds 5-14, 16, 17 and 20, fluorescence
intensity of the tryptophan residue (Trp-212) present in BSA decreased with a concomitant
increase in the fluorescence intensity through an isoemissive point within range of 339 nm
and 357 nm (Figure 3, Table 3). This indicates an efficient energy transfer from the
tryptophan residue (Trp-212) present in BSA to compounds 5-14, 16, 17 and 20 (Figures
S64-S67). The overlap of absorption spectrum of compounds 6, 16, 17 and 20 and the
emission spectrum of BSA are shown in Figure 4. It has been observed that distance (r)
between the Trp residue (as donor) and the interacting compound (as acceptor) is in the range
of 4−8 nm. According to Valuer et. al., the maximum academic value for Ro should be < 10
nm and r should be less than 8 nm [33]. In the present study, both Ro and r values are well
within the permissible limits. These results indicated that the energy transfer from BSA to
compounds occur with high probability.
Figure 4
Table 4

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3. Conclusions
In summary, a series of novel substituted triazine-benzimidazole analogs have been
1
synthesized and characterized by H and 13C NMR. These compounds were evaluated for
growth inhibition properties against a panel of 60 human cancer cell lines, and their GI50, TGI
and LC50 values have been determined. Four compounds (6, 16, 17 and 20) have been
identified with GI50’s in the nanomolar range for most of the cell lines. These compounds
also strongly interacted with BSA, as determined by UV-Visible and fluorescence
spectroscopy. The binding constant values suggest that triazine-benzimidazole binds to the
high affinity binding sites within albumin. From Forster non-radiative energy transfer
equations, it has been found that the distance of compounds from BSA is in the range of 4-8
nm which predicts the possibility of energy transfer. The biological significance of this work
is evident since the anticancer activity and the interaction of triazine-benzimidazole
conjugates with albumin was not characterized earlier. Thus, triazine-benzimidazole studies
clearly demonstrate that hybrids with heterocyclic hydrophobic groups are beneficial in
biological and anticancer activity. These preliminary biological screening results offer
encouragement and further mechanistic studies of these types of analogues are in progress.
4. Experimental section
4.1. Chemistry
All materials were obtained from commercial suppliers and used without further
purification unless otherwise noted. Melting points were determined in open capillaries and
were uncorrected. Reactions were monitored by thin-layer chromatography (TLC) carried out
1
13
on silica gel plates (GF 254) using UV light as visualizing agents. H NMR and C NMR
13
spectra were recorded on Jeol-400 (1H, 400 MHz; C, 100 MHz) spectrometer at ambient
temperature, using CDCl3, DMSO-d6 and trifluoroacetic acid (TFA) as solvents. Chemical
shifts are reported in parts per million (ppm) with TMS as an internal reference and J values
are given in Hz. Mass spectrometric data were recorded at Waters Micromass Q-Tof Micro.
Elemental analysis was done with Thermo Scientific (Flash 2000) analyzer. Hexane:ethyl
acetate and chloroform:methanol were the adopted solvent systems. UV-Visible studies were
carried out Shimadzu-2400 PC spectrometer. The fluorescence spectra were determined on a
Varian Cary Eclipse fluorescence spectrometer.
4.2.
Synthesis of 4,6-dichloro-2-morpholin-4-yl-[1,3,5]triazine (2)

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To a stirred solution of cyanuric chloride (10 g, 0.054 mol) in anhydrous THF (150 mL),
0
morpholine (5.65 g, 0.065 mol) was added at 0-5 C. To this mixture, 10% NaHCO3 was
added and stirred at the same temperature for 6 h. The resulted reaction mixture was then
poured into crushed ice, filtered, dried and column chromatographed on silica gel in hexane :
ethylacetate to afford the desired product 4,6-dichloro-2-morpholin-4-yl-[1,3,5]triazine (2) as
white solid; (11.33 g, 89%); mp: 277-279 0C [34].
4.3.
Synthesis of 4-(1H-benzimidazol-2-yl)-phenylamine (3)
A mixture of 4-aminobenzoic acid (5 g, 5.78 mmol) and o-phenylenediamine (3.9 g, 3.68
mmol) were stirred in a syrupy polyphosphoric acid (12.5 gm) at 200 0C for 5 h. The reaction
was monitored by TLC. The reaction mixture was cooled and poured into crushed ice. The
white precipitate was then stirred in cold water. Ammonium hydroxide solution was added
until the pH 7.0 was achieved. The resulting solid was filtered and washed several times with
methanol and column chromatographed on silica gel in hexane : ethylacetate to afford the
desired product 4-(1H-benzimidazol-2-yl)-phenylamine (3) as white solid; (82%); mp: 207-
o
1
209 C; H NMR (400 MHz, DMSO-d6): δ = 7.96 (d, 2H, J = 8.72 Hz, ArH), 7.55-7.51 (m,
13
2H, ArH), 7.16-7.14 (m, 2H, ArH), 6.75 (d, 2H, J = 8.24 Hz, ArH), 4.45 (bs, 2H, NH2); C
NMR (100 MHz, CDCl3 + DMSO-d6): δ = 152.2, 148.4, 127.5, 120.9, 118.6, 113.8 (ArC);
MS(ESI), m/z: 209.2 (M++1).
4.4.
Synthesis
of
[4-(3H-benzimidazol-5-yl)-phenyl]-(6-chloro-2-morpholin-4-yl-
[1,3,5]triazin-2-yl)-amine (4)
To a stirred solution of 4,6-dichloro-2-morpholin-4-yl-[1,3,5]triazine (2) (10 g, 0.042 mol) in
anhydrous THF (150 mL), 4-(1H-benzimidazol-2-yl)-phenylamine (3) (10.67 g, 0.051 mol)
was added at room temperature. To this mixture, 10% K2CO3 was added and stirred for 24 h.
The resulted reaction mixture was then poured into crushed ice, filtered, dried and column
chromatographed on silica gel using hexane : ethylacetate to afford the desired product [4-
(3H-benzimidazol-5-yl)-phenyl]-(6-chloro-2-morpholin-4-yl-[1,3,5]triazin-2-yl)-amine
(4)
as white solid; 84%; mp: 277-279 oC; 1H NMR (400 MHz, CDCl3 + DMSO-d6): δ = 9.94 (bs,
1H, NH), 8.14 (d, 2H, J = 8.68 Hz, ArH), 7.81 (d, 2H, J = 8.72 Hz, ArH), 7.62-7.56 (m, 2H,
ArH), 7.21-7.18 (m, 2H, ArH), 3.87-3.86 (m, 4H, mor-CH2), 3.77-3.75 (m, 4H, mor-CH2);
13C NMR (100 MHz, CDCl3): δ = 152.9, 142.8, 135.6, 129.1, 128.1, 126.2, 109.4 (ArC), 66.7
(O-CH2), 47.3 (N-CH2); MS(ESI), m/z: 409.5 (M++2); Anal. Calcd for C20H18ClN7O: C,
58.90; H, 4.45; N, 24.04, Found: C, 58.99; H, 4.41; N, 24.20.

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4.5.
General procedure for the synthesis of compounds 5-15
To a stirred solution of [4-(3H-benzimidazol-5-yl)-phenyl]-(6-chloro-2-morpholin-4-yl-
[1,3,5]triazine-2,4-diamine (4) (0.200 g, 0.463 mmol) in 1,4-dioxane (20 mL), amine (0.556
o
mmol) and potassium carbonate (0.095g, 0.695 mmol) were added and heated at 110 C for
8-12 h. Extracted the reaction mixture with chloroform, dried over Na2SO4, filtered and
concentrated to get crude product which was then purified through column chromatography
using chloroform:methanol (50:1) as eluents to give compounds 5-15.
4.5.1. [4-(3H-Benzimidazol-5-yl)-phenyl]-(2,6-di-morpholin-4-yl-[1,3,5]triazin-2-yl)-amine
(5)
White solid; yield: 78%; mp: 268-270 oC; 1H NMR (400 MHz, CDCl3): δ = 8.00 (d, 2H, J =
7.76 Hz, ArH), 7.70 (d, 3H, J = 8.24 Hz, ArH), 7.27-7.25 (m, 3H, ArH), 6.87 (bs, 1H, NH),
3.81-3.76 (m, 8H, mor-CH2), 3.74-3.70 (m, 8H, mor-CH2); 13C NMR (100 MHz, CDCl3): δ =
165.1, 151.6, 135.8, 129.2, 128.2, 128.0, 125.7, 118.9, 115.5, 101.1 (ArC), 66.8 (O-CH2),
43.6 (N-CH2); MS(ESI), m/z: 481.3 (M++23); Anal. Calcd for C24H26N8O2: C, 62.87; H,
5.72; N, 24.44, Found: C, 62.98; H, 5.67; N, 24.55.
4.5.2. [4-(3H-Benzimidazol-5-yl)-phenyl]-(2-morpholin-4-yl-6-piperidin-1-yl-[1,3,5]triazin-
2-yl)-amine (6)
White solid; yield: 72%; mp: 271-273 oC; 1H NMR (400 MHz, CDCl3): δ = 8.00 (d, 2H, J =
8.72 Hz, ArH), 7.72 (d, 2H, J = 8.72 Hz, ArH), 7.62 (s, 1H, ArH), 7.26-7.24 (m, 3H, ArH),
6.88 (bs, 1H, NH), 3.79 (t, 4H, J = 5.42 Hz, mor-CH2), 3.75-3.68 (m, 8H, mor-CH2, pip-CH2),
13
1.68-1.59 (m, 6H, pip-CH2); C NMR (100 MHz, CDCl3): δ = 152.3, 143.0, 135.9, 134.6,
131.4, 129.0, 127.9, 126.2, 116.1, 110.5, 109.0, (ArC), 67.0 (O-CH2), 44.2 (N-CH2), 43.7 (N-
CH2), 25.9 (CH2), 25.0 (CH2); MS(ESI), m/z: 479.5 (M++23); Anal. Calcd for C25H28N8O: C,
65.77; H, 6.18; N, 24.54, Found: C, 65.65; H, 6.14; N, 24.62.
4.5.3. [4-(3H-Benzimidazol-5-yl)-phenyl]-(2-morpholin-4-yl-6-pyrrolidin-1-yl-
[1,3,5]triazin-2-yl)-amine (7)
White solid; yield: 76%; mp: 277-279 oC; 1H NMR (400 MHz, CDCl3): δ = 7.99 (d, 2H, J =
8.68 Hz, ArH), 7.75 (d, 2H, J = 8.68 Hz, ArH), 7.61 (bs, 1H, NH), 7.25-7.23 (m, 4H, ArH),
6.94 (bs, 1H, NH), 3.82-3.79 (m, 4H, mor-CH2), 3.74-3.72 (m, 4H, mor-CH2), 3.57 (t, 4H, J
= 5.04 Hz, pyrr-CH2), 1.96-1.94 (m, 4H, pyrr-CH2); 13C NMR (100 MHz, CDCl3): δ = 152.2,
143.0, 136.0, 134.8, 129.0, 127.9, 126.2, 115.8, 110.3, 109.0 (ArC), 67.0 (O-CH2), 46.1 (N-

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CH2), 43.7 (N-CH2), 25.4 (CH2); MS(ESI), m/z: 442.3 (M+); Anal. Calcd for C24H26N8O: C,
65.14; H, 5.92; N, 25.32, Found: C, 65.25; H, 5.88; N, 25.39.
4.5.4. [4-(3H-Benzimidazol-5-yl)-phenyl]-[6-(4-methyl-piperazin-1-yl)-2-morpholin-4-yl-
[1,3,5]triazin-2-yl]-amine (8)
White solid; yield: 77%; mp: 282-284 oC; 1H NMR (400 MHz, CDCl3): δ = 8.01 (d, 2H, J =
8.72 Hz, ArH), 7.70 (d, 2H, J = 8.72 Hz, ArH), 7.63 (bs, 1H, NH), 7.27-7.24 (m, 4H, ArH),
6.87 (bs, 1H, NH), 3.84 (t, 4H, J = 4.12 Hz, mor-CH2), 3.80-3.78 (m, 4H, mor-CH2), 3.75-
3.72 (m, 4H, piperazine-CH2), 2.46 (t, 4H, J = 4.60 Hz, piperazine-CH2), 2.35 (s, 3H, N-
CH3); 13C NMR (100 MHz, CDCl3): δ = 138.2, 135.9, 135.8, 135.1, 129.1, 127.9, 125.9,
118.9, 115.2, 100.8 (ArC), 66.8 (O-CH2), 46.8 (N-CH2), 44.2 (N-CH2), 43.6 (N-CH2), 14.0
(N-CH3); MS(ESI), m/z: 471.5 (M+); Anal. Calcd for C25H29N9O: C, 63.68; H, 6.20; N,
26.73, Found: C, 63.79; H, 6.15; N, 26.66.
4.5.5. N-[4-(3H-Benzimidazol-5-yl)-phenyl]-2-morpholin-4-yl-N'-(6-morpholin-4-yl-ethyl)-
[1,3,5]triazine-4,6-diamine (9)
o
1
White solid; yield: 69%; mp: 289-291 C; H NMR (400 MHz, CDCl3): δ = 8.05 (s, 1H,
ArH), 7.30 (t, 4H, J = 7.36 Hz, ArH, NH), 7.10 (d, 2H, J = 8.24 Hz, ArH), 7.05 (d, 2H, J =
11.36 Hz, ArH), 3.82-3.80 (m, 8H, mor-CH2), 3.72-3.69 (m, 8H, mor-CH2), 3.51-3.49 (m,
2H, N-CH2), 2.20-2.17 (m, 2H, N-CH2); 13C NMR (100 MHz, CDCl3): δ = 166.0, 164.6,
152.5, 142.9, 135.8, 129.1, 128.0, 126.3, 122.1, 121.7, 115.5, 115.2, 109.1 (ArC), 66.9 (O-
CH2), 57.2 (N-CH2), 53.4 (N-CH2), 47.2 (N-CH2), 37.1 (N-CH2); MS(ESI), m/z: 524.3
(M++23); Anal. Calcd for C26H31N9O2: C, 62.26; H, 6.23; N, 25.13, Found: C, 62.30; H, 6.18;
N, 25.25.
4.5.6. 2-(4-{4-[4-(3H-Benzimidazol-5-yl)-phenylamino]-2-morpholin-4-yl-[1,3,5]triazin-6-
yl}-piperazin-1-yl)-ethanol (10)
White solid; yield: 68%; mp: 287-289 oC; 1H NMR (400 MHz, CDCl3): δ = 7.60 (d, 1H, J =
8.72 Hz, ArH), 7.33-7.27 (m, 3H, ArH), 7.17 (s, 1H, ArH), 7.08 (d, 1H, J = 8.72 Hz, ArH),
7.03 (d, 2H, J = 6.40 Hz, ArH), 3.67-3.57 (m, 12H, mor-CH2, piperazine-CH2), 3.47 (d, 2H, J
= 8.28 Hz, O-CH2), 2.43-2.42 (m, 2H, N-CH2), 2.15-2.13 (m, 4H, piperazine-CH2); 13C NMR
(100 MHz, CDCl3): δ = 151.7, 138.4, 135.9, 135.8, 134.7, 129.1, 127.9, 125.9, 118.9, 115.7,
115.5 (ArC), 66.9 (O-CH2), 66.7 (O-CH2), 57.3 (N-CH2), 53.4 (N-CH2), 43.6 (N-CH2), 36.9
(N-CH2); MS(ESI), m/z: 501.3 (M+); Anal. Calcd for C26H31N9O2: C, 62.26; H, 6.23; N,
25.13, Found: C, 62.15; H, 6.18; N, 25.24.

ACCEPTED MANUSCRIPT
4.5.7. N-(6-Amino-ethyl)-N'-[4-(3H-benzimidazol-5-yl)-phenyl]-2-morpholin-4-yl-
[1,3,5]triazine-4,6-diamine (11)
White solid; yield: 73%; mp: 265-267 oC; 1H NMR (400 MHz, CDCl3): δ = 7.95 (d, 2H, J =
7.76 Hz, ArH), 7.62-7.59 (m, 4H, ArH, NH), 7.25-7.22 (m, 3H, ArH), 3.75-3.73 (m, 4H,
mor-CH2), 3.71-3.67 (m, 4H, mor-CH2), 3.46 (t, 2H, J = 5.96 Hz, N-CH2), 2.91 (t, 2H, J =
5.48 Hz, N-CH2); 13C NMR (100 MHz, CDCl3): δ = 152.4, 143.0, 135.9, 134.2, 131.7, 129.1,
128.0, 126.2, 109.1 (ArC), 66.9 (O-CH2), 47.2 (N-CH2), 43.7 (N-CH2), 41.8 (N-CH2);
MS(ESI), m/z: 431.3 (M+); Anal. Calcd for C22H25N9O: C, 61.24; H, 5.84; N, 29.21, Found:
C, 61.39; H, 5.80; N, 29.14.
4.5.8. 2-{4-[4-(3H-Benzimidazol-5-yl)-phenylamino]-2-morpholin-4-yl-[1,3,5]triazin-6-
ylamino}-ethanol (12)
o
1
White solid; yield: 71%; mp: 262-264 C; H NMR (400 MHz, CDCl3): δ = 8.05 (s, 1H,
ArH), 7.31 (d, 3H, J = 7.36 Hz, ArH), 7.24-7.22 (m, 1H, ArH), 7.11 (d, 1H, J = 8.24 Hz,
ArH), 7.05 (d, 2H, J = 6.88 Hz, ArH), 3.79-3.72 (m, 8H, mor-CH2), 3.49 (t, 2H, J = 5.48 Hz,
13
O-CH2), 2.91-2.88 (m, 2H, N-CH2); C NMR (100 MHz, CDCl3): δ = 152.5, 142.8, 135.8,
133.9, 131.8, 129.1, 128.0, 126.2, 116.6, 111.4, 109.1 (ArC), 66.8 (O-CH2), 63.8 (O-CH2),
47.2 (N-CH2), 43.7 (N-CH2); MS(ESI), m/z: 455.9 (M++23); Anal. Calcd for C22H24N8O2: C,
61.10; H, 5.59; N, 25.91, Found: C, 61.01; H, 5.55; N, 25.99.
4.5.9. N-[4-(3H-Benzimidazol-5-yl)-phenyl]-N'-benzyl-2-morpholin-4-yl-[1,3,5]triazine-4,6-
diamine (13)
White solid; yield: 66%; mp: 275-277 oC; 1H NMR (400 MHz, CDCl3): δ = 8.04 (d, 1H, J =
1.80 Hz, ArH), 7.30-7.27 (m, 4H, ArH), 7.24 (d, 1H, J = 1.80 Hz, ArH), 7.22 (d, 1H, J = 2.28
Hz, ArH), 7.11 (d, 2H, J = 8.72 Hz, ArH), 7.05 (d, 2H, J = 1.84 Hz, ArH), 7.03 (d, 2H, J =
4.12 Hz, ArH), 5.28 (s, 2H, N-CH2), 3.72 (t, 8H, J = 8.72 Hz, mor-CH2); 13C NMR (100
MHz, CDCl3): δ = 151.9, 138.8, 135.8, 134.9, 134.0, 129.1, 128.0, 126.0, 122.1, 119.1, 115.5,
115.3, 101.7 (ArC), 66.7 (O-CH2), 46.9 (N-CH2), 43.7 (N-CH2); MS(ESI), m/z: 478.3 (M+);
Anal. Calcd for C27H26N8O: C, 67.77; H, 5.48; N, 23.42, Found: C, 67.71; H, 5.43; N, 23.49.
4.5.10. N-[4-(3H-Benzimidazol-5-yl)-phenyl]-N'-cyclohexyl-2-morpholin-4-yl-[1,3,5]triazine-
4,6-diamine (14)
White solid; yield: 68%; mp: 283-285 oC; 1H NMR (400 MHz, CDCl3): δ = 8.00 (d, 2H, J =
7.76 Hz, ArH), 7.76 (d, 1H, J = 6.88 Hz, ArH), 7.67-7.62 (m, 2H, ArH), 7.25-7.23 (m, 2H,

ACCEPTED MANUSCRIPT
ArH), 6.95 (s, 1H, ArH), 4.95 (m, 1H, N-CH), 3.79-3.73 (m, 8H, mor-CH2), 2.05-2.02 (m,
2H, CH2), 1.77-1.74 (m, 2H, CH2), 1.66-1.64 (m, 2H, CH2), 1.41-1.38 (m, 2H, CH2), 1.25-
1.19 (m, 2H, CH2); 13C NMR (100 MHz, CDCl3): δ = 165.8, 164.4, 152.0, 151.9, 135.9,
135.7, 129.0, 127.9, 126.3, 126.1, 119.0, 115.5, 115.3 (ArC), 66.9 (O-CH2), 57.2 (N-CH),
53.4 (N-CH2), 37.1 (CH2), 32.0 (CH2), 22.7 (CH2); MS(ESI), m/z: 471.3 (M++1); Anal. Calcd
for C26H30N8O: C, 66.36; H, 6.43; N, 23.81, Found: C, 66.48; H, 6.38; N, 23.89.
4.5.11. [4-(3H-Benzimidazol-5-yl)-phenyl]-(6-hydrazino-2-morpholin-4-yl-[1,3,5]triazin-4-
yl)-amine (15)
White solid; yield: 64%; mp: 296-298 oC; 1H NMR (400 MHz, CDCl3): δ = 7.67 (d, 1H, J =
8.60 Hz, ArH), 7.61 (d, 1H, J = 8.72 Hz, ArH), 7.48 (bs, 1H, NH), 7.25-7.23 (m, 2H, ArH),
7.11-6.98 (m, 4H, ArH), 3.67-3.65 (m, 4H, mor-CH2), 3.50-3.47 (m, 4H, mor-CH2); 13C
NMR (100 MHz, CDCl3): δ = 152.4, 139.4, 135.6, 132.8, 129.2, 128.1, 125.7, 119.2, 116.2,
102.1 (ArC), 66.6 (O-CH2), 43.9 (N-CH2); MS(ESI), m/z: 403.7 (M+); Anal. Calcd for
C20H21N9O: C, 59.54; H, 5.25; N, 31.25, Found: C, 59.64; H, 5.21; N, 31.28.
4.6.
General procedure for the synthesis of compounds 16-21
To a stirred solution of [4-(3H-benzimidazol-5-yl)-phenyl]-(6-chloro-2-morpholin-4-yl-
[1,3,5]triazin-2-yl)-amine (4) (0.200 g, 0.490 mmol) in 1,4-dioxane (20 mL), arylboronic
acids (0.556 mmol), Pd(PPh3)4 (10 mol%) and potassium carbonate (0.101g, 0.736 mmol)
were added and refluxed for 8-10 h in an inert atmosphere. After the completion of reaction,
extracted the reaction mixture with chloroform, dried over Na2SO4, filtered and concentrated
to get crude product which was purified through column chromatography using
chloroform:methanol (50:1) as eluents to give compounds 16-21.
4.6.1. [4-(3H-Benzimidazol-5-yl)-phenyl]-(2-morpholin-4-yl-6-phenyl-[1,3,5]triazin-2-yl)-
amine (16)
White solid; yield: 72%; mp: 267-269 oC; 1H NMR (400 MHz, CDCl3): δ = 8.06 (d, 1H, J =
8.72 Hz, ArH), 8.00 (d, 1H, J = 4.68 Hz, ArH), 7.85 (d, 1H, J = 8.24 Hz, ArH), 7.77 (d, 1H, J
= 8.96 Hz, ArH), 7.72 (d, 2H, J = 8.72 Hz, ArH), 7.62 (bs, 1H, NH), 7.54-7.52 (m, 3H, ArH),
13
7.08 (t, 2H, J = 8.72 Hz, ArH), 6.77-6.70 (m, 2H, ArH), 3.99-3.95 (m, 8H, mor-CH2); C
NMR (100 MHz, CDCl3): δ = 152.0, 137.6, 135.8, 134.0, 132.1, 129.7, 129.2, 128.6, 128.5,
128.0, 125.8, 119.2, 115.7, 101.5 (ArC), 66.8 (O-CH2), 43.7 (N-CH2); MS(ESI), m/z: 472.6
(M++23); Anal. Calcd for C26H23N7O: C, 69.47; H, 5.16; N, 21.81, Found: C, 69.64; H, 5.12;
N, 21.98.

ACCEPTED MANUSCRIPT
4.6.2. [4-(3H-Benzimidazol-5-yl)-phenyl]-[6-(4-fluoro-phenyl)-2-morpholin-4-yl-
[1,3,5]triazin-2-yl]-amine (17)
White solid; yield: 71%; mp: 279-281 oC; 1H NMR (400 MHz, CDCl3): δ = 8.34 (d, 2H, J =
7.32 Hz, ArH), 8.03 (bs, 1H, NH), 7.67 (d, 1H, J = 8.28 Hz, ArH), 7.48 (d, 1H, J = 7.32 Hz,
ArH), 7.40-7.37 (m, 2H, ArH), 7.35-7.29 (m, 3H, ArH), 7.16 (d, 1H, J = 2.32 Hz, ArH), 7.07
13
(d, 2H, J = 6.40 Hz, ArH), 3.98-3.61 (m, 8H, mor-CH2); C NMR (100 MHz, CDCl3): δ =
151.9, 138.8, 135.8, 134.9, 134.0, 129.1, 128.0, 126.0, 122.1, 119.1, 115.5, 115.3, 101.7
(ArC), 66.7 (O-CH2), 43.7 (N-CH2); MS(ESI), m/z: 467.3 (M+); Anal. Calcd for
C26H22FN7O: C, 66.80; H, 4.74; N, 20.97, Found: C, 66.85; H, 4.71; N, 21.05.
4.6.3. [4-(3H-Benzimidazol-5-yl)-phenyl]-(6-furan-2-yl-2-morpholin-4-yl-[1,3,5]triazin-4-
yl)-amine (18)
White solid; yield: 66%; mp: 282-284 oC; 1H NMR (400 MHz, CDCl3): δ = 8.00 (d, 2H, J =
4.72 Hz, ArH), 7.83 (bs, 1H, NH), 7.72 (d, 2H, J = 8.24 Hz, ArH), 7.52-7.48 (m, 3H, ArH),
7.07 (t, 3H, J = 8.68 Hz, ArH), 6.96 (s, 1H, ArH), 6.80 (bs, 1H, NH), 3.77-3.69 (m, 8H, mor-
CH2); 13C NMR (100 MHz, CDCl3): δ = 152.9, 142.8, 135.6, 132.5, 129.1, 128.1, 126.2,
109.4 (ArC), 66.7 (O-CH2), 44.1 (N-CH2); MS(ESI), m/z: 439.2 (M+); Anal. Calcd for
C24H21N7O2: C, 65.59; H, 4.82; N, 22.31, Found: C, 65.86; H, 4.87; N, 22.39.
4.6.4. 4-(3H-Benzimidazol-5-yl)-phenyl]-(2-morpholin-4-yl-6-thiophen-2-yl-[1,3,5]triazin-
2-yl)-amine (19)
White solid; yield: 67%; mp: 288-290 oC; 1H NMR (400 MHz, CDCl3): δ = 7.50 (q, 3H, J =
5.04 Hz, ArH), 7.37-7.31 (m, 3H, ArH), 7.14 (d, 2H, J = 5.96 Hz, ArH), 7.03 (t, 3H, J = 8.72
Hz, ArH), 6.86 (bs, 1H, NH), 3.79-3.78 (m, 4H, mor-CH2), 3.75-3.74 (m, 4H, mor-CH2); 13C
NMR (100 MHz, CDCl3): δ = 152.6, 143.0, 135.8, 135.1, 133.6, 132.0, 129.1, 128.0, 126.2,
121.7, 115.5, 115.3, 109.1 (ArC), 66.9 (O-CH2), 43.8 (N-CH2); MS(ESI), m/z: 478.3
(M++23); Anal. Calcd for C24H21N7OS: C, 63.28; H, 4.65; N, 21.52; S, 7.04, Found: C, 63.15;
H, 4.61; N, 21.59; S, 7.08.
4.6.5. [4-(3H-Benzimidazol-5-yl)-phenyl]-[6-(4-chloro-phenyl)-2-morpholin-4-yl-
[1,3,5]triazin-2-yl]-amine (20)
White solid; yield: 68%; mp: 276-278 oC; 1H NMR (400 MHz, CDCl3): δ = 8.35 (d, 2H, J =
5.48 Hz, ArH), 8.01 (bs, 1H, NH), 7.67 (d, 1H, J = 8.24 Hz, ArH), 7.33-7.31 (m, 4H, ArH),
7.16 (d, 2H, J = 8.68 Hz, ArH), 7.07 (d, 3H, J = 5.96 Hz, ArH), 3.99-3.73 (m, 8H, mor-CH2);
13C NMR (100 MHz, CDCl3): δ = 152.4, 139.4, 135.6, 132.8, 129.2, 128.1, 125.7, 119.2,

ACCEPTED MANUSCRIPT
116.2, 102.1 (ArC), 66.6 (O-CH2), 43.9 (N-CH2); MS(ESI), m/z: 484.3 (M++1); Anal. Calcd
for C26H22ClN7O: C, 64.53; H, 4.58; N, 20.26, Found: C, 64.64; H, 4.41; N, 20.31.
4.6.6. [4-(3H-Benzimidazol-5-yl)-phenyl]-[6-(4-trifluoromethyl-phenyl)-2-morpholin-4-yl-
[1,3,5]triazin-2-yl]-amine (21)
o
1
White solid; yield: 65%; mp: 279-281 C; H NMR (400 MHz, CDCl3): δ = 8.03 (s, 1H,
ArH), 7.50-7.46 (m, 2H, ArH), 7.31-7.28 (m, 2H, ArH), 7.24-7.22 (m, 2H, ArH), 7.13 (d, 1H,
J = 8.72 Hz, ArH), 7.06 (d, 2H, J = 6.88 Hz, ArH), 6.99-6.97 (m, 2H, ArH), 6.86 (bs, 1H,
NH), 3.81-3.78 (m, 4H, mor-CH2), 3.74-3.71 (m, 4H, mor-CH2); 13C NMR (100 MHz,
CDCl3): δ = 152.0, 138.7, 136.8, 135.8, 134.1, 131.6, 129.6, 129.2, 128.3, 128.0, 125.9,
119.1, 115.6, 101.4 (ArC), 66.8 (O-CH2), 43.7 (N-CH2); MS(ESI), m/z: 517.3 (M+); Anal.
Calcd for C27H22F3N7O: C, 62.66; H, 4.28; N, 18.95, Found: C, 67.59; H, 4.18; N, 18.85.
4.7. Procedure for in vitro anticancer screening
The human tumor cell lines of cancer screening were grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. Cells were inoculated in 96 well
plates in 100 ꢁL per well at plating densities ranging from 5,000 to 40,000 cells/well that
depends upon the doubling time of individual cell lines. The microtiter plates were then
°
incubated at 37 C, 95% air, 5% CO2, and 100% relative humidity for 24 h. Two plates of
each cell line were then fixed in situ with TCA. Experimental drugs were dissolved in DMSO
at 400-fold the desired final maximum test concentration and stored frozen prior to use. At
the time of drug addition, an aliquot of frozen concentrate was used and diluted to two times
the desired final maximum test concentration with complete medium containing 50 µg/ml
gentamicin. Additional four, 10-fold or ½ log serial dilutions were made to give total of five
drug concentrations plus control. Aliquots of 100 µL of these drug dilutions were added to
°
the appropriate microtiter wells. Following the addition, plates were incubated at 37 C, 5%
CO2, 95% air, and 100% relative humidity for 48 h. For adherent cells, the assay was
terminated by the addition of cold TCA. Cells were fixed in situ by the addition of 50 µL of
°
cold 50% (w/v) TCA and incubated at 4 C for 60 min. The plates were washed five times
with tap water by discarding the supernatant and air dried. Sulforhodamine B (SRB) solution
(100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well followed by incubation of
plates for 10 min at room temperature. After staining, unbound dye was removed by washing
five times with 1% acetic acid and the plates were air dried before subsequent solubilization
with 10 mM trizma base. The absorbance was read at a wavelength of 515 nm on an

ACCEPTED MANUSCRIPT
automated plate reader. With seven absorbance measurements [time zero (Tz), control growth
(C), and test growth in the presence of drug at five concentration levels (Ti)], percentage
growth was calculated at each of the drug concentration levels. Percentage growth inhibition
was calculated as:
[(Ti -Tz)/(C - Tz)] × 100 for concentrations for which Ti ≥ Tz; [(Ti -Tz)/Tz] × 100 for
concentrations for which Ti < Tz.
Three dose response parameters were calculated for each experimental agent. Growth
inhibition of 50% (GI50) was calculated from [(Ti -Tz)/(C - Tz)] × 100 = 50. Drug
concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. LC50
was calculated from [(Ti-Tz)/Tz]×100 = 50
4.8.
Procedure for BSA screening
4.8.1. Materials and methods
The stock solution of BSA (Sigma Chemical Co., USA) was prepared by dissolving an
appropriate amount of solid BSA in 0.1 M phosphate buffer at pH 7.4 and stored at 0−4 °C in
the dark. Stock solutions of compounds 5-21 (10−3 mol L−1) were prepared in DMSO. All
stock solutions were diluted to the required concentrations with phosphate buffer (pH 7.4).
All other reagents were of analytical reagent grade.
4.8.2. UV-Visible absorption and fluorescence spectra
UV-visible absorption and fluorescence emission spectra were measured with a 1 cm quartz
cell. All the spectra were recorded at ambient temperature (300 K). UV-Vis spectra were
recorded on Shimadzu-2400 PC spectrometer. The wavelength of the spectra was between
200 and 800 nm. All the UV-visible spectra were recorded after equilibration of solution for 5
min. Emission spectra were recorded with Varian Cary Eclipse fluorescence spectrometer.
The excitation and emission wavelengths for BSA were 280 nm and 351 nm with a slit width
of 10 nm. Very dilute solutions of BSA and compounds 5-21 were used to avoid inner filter
effect. The titration experiments were performed by varying the concentration of compounds
5-21 and keeping the BSA concentration (10 µM).
Binding constant K was estimated from absorption and fluorescence titration data using the
Benesi–Hildebrand equation. K was determined from the ratio of intercept to slope obtained
from the linear fit of the plot of 1/(A0 − A) vs. 1/[compound] and 1/(F0 − F) vs.

ACCEPTED MANUSCRIPT
1/[compound], where A0 and A are the absorption intensities and F0 and F are the emission
intensities of the BSA in the absence and presence of compounds, respectively.
1/(A0 − A) = 1/(A0 − Afc) + {1/[K(A0 − Afc)]}[ligand]
To determine the binding number of triazine-benzimidazole analogues with BSA, the double-
logarithmic equation was used:
log[(F0-F)/F] = log K + n log [Q]
where, F0 and F are the emission intensities of BSA in the absence and presence of triazine-
benzimidazole analogues, respectively. A plot of log[(F0-F)/F] vs. log[Q] will produce a
straight line; whose slope is equal to n. The number of binding sites n as calculated from the
plot is approximately equal to 1 for BSA and it is in good agreement with the literature
reports.
Acknowledgments
KP thanks the Department of Science and Technology, New Delhi (EMR/2014/000669). PS
is grateful for a DST/Inspire Fellowship (Fellow Code-IF110542). NCI, NIH, USA was
greatly acknowledged for anticancer activities.
Abbreviations
BSA, bovine serum albumin; Trp-134 and Trp-212, tryptophan residues; NCI, National
Cancer Institution, CNS, central nervous system; GI %, percentage growth inhibition; GI50,
50 % growth inhibition; TGI, total growth inhibition; LC50, 50 % lethal concentration; MG-
MID, mean graph midpoints; Trp, tryptophan; Tyr, tyrosine; Phe, phenylalanine; UV-Vis.,
ultraviolet visible; FRET, fluorescence resonance energy transfer; K, binding constant; n,
number of binding site, E, efficiency of energy transfer; R0, critical distance; K2, orientation
factor; n, refractive index; Φ, quantum yield; J, degree of spectral overlap; THF,
tetrahydrofuran; TLC, thin layer chromatography.
Notes and References
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[5] (a) P. Singla, V. Luxami, K. Paul, Bioorg. Med. Chem. 23 (2015) 1691-1700. (b) P.

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Singla, V. Luxami, K. Paul, Bioorg. Med. Chem. Lett. 26 (2016), 518-523.
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McCarter, T.S. Miguel, L. Zalameda, J. Jiang, R. Subramanian, E.L. Mullady, S.
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ACCEPTED MANUSCRIPT
Figures Captions
Figure 1. Triazines possessing anticancer activity.
Figure 2. Absorption spectral changes of BSA (10 ꢀM) in phosphate buffer upon addition of
compounds 6 (a), 16 (b), 17 (c) and 20 (d) at 0-20 ꢀM; Insets indicate Benesi–Hildebrand plot
of 1/[A0-A] vs. 1/[compounds] for binding of BSA with compounds 6 (a), 16 (b), 17 (c) and
20 (d) through absorption spectral titrations.
Figure 3. Fluorescence spectral changes of BSA (10 ꢀM) in phosphate buffer upon addition
of 6 (a), 16 (b), 17 (c) and 20 (d) at 0-20 ꢀM. Insets indicate Benesi–Hildebrand plot of 1/[F0-
F] vs. 1/[compounds] for binding of BSA with compounds 6 (a), 16 (b), 17 (c) and 20 (d)
through emission spectral titrations.
Figure 4. Overlap of the fluorescence emission spectra of BSA with UV-Visible absorption
spectra of compounds 6 (a), 16 (b), 17 (c) and 20 (d).
Scheme 1. Synthesis of triazine-benzmidazoles
Table 1. Effects of 10 ꢀM of compounds 6, 16, 17 and 20 on cell proliferation in a subpanel
tumor cell lines.
Table 2. Compounds 6, 16, 17 and 20 with median growth inhibitory (GI50, µM), total
growth inhibitory (TGI, µM) and median lethal concentrations (LC50, µM) of in vitro tumor
cell lines at five different concentrations viz., 10-4, 10-5, 10-6, 10-7 and 10-8 M
Table 3. Photophysical properties and binding constants of BSA with compounds 5-14, 16,
17 and 20.
Table 4. Forster energy transfer parameters for BSA and triazine-benzimidazoles (5-14, 16,
17 and 20).
O
NH2
N
NH2
N
NH2
N
N
N
NMe2
Br
Me
Cl
N
N
N
N
N
O
N
N
N
Cl
N
N
H2N
N
N
N
N
H
N
O
N
Me2N
NMe2
H
Cl
O
N
N
H
NMe2
H
Irsogladine
Altretamine
CT32228
Gedatolisib
HL010183
O
N
O
N
O
N
N
N
S
N
H
N
N
N
N
N
N
S
HN
O
Cl
N
N
N
N
N
N
NH
R2R1N
N
N
H
O
O
PZ-39
SEF19
5-21

ACCEPTED MANUSCRIPT
Figure 1. Triazines possessing anticancer activity.
NR1R2
=
5; morpholine
6
O
N
; piperidine
O
N
O
N
N
7; pyrrolidine
H2N
Cl
N
8
9
; N-methylpiperazine
N
N
H
HN
O
; 2-aminoethylmorpholine
3
N
N
HNR1R2, K2CO3
N
N
N
N
N
N
N
N
H
H
10; 2-hydroxyethylpiperazine
11
Cl
N
Cl
NaHCO3, THF
0-5 °C
1,4-dioxane
110 0C
R2R1N
N
N
K2CO3
,
Cl
N
N
H
; 1,2-ethylenediamine
12;1,2-ethanolamine
Cl
N
Cl
H
THF, rt
1
2
5-15
4
13
14
; benzylamine
; cyclohexylamine
K2CO3,
1,4-dioxane,
reflux
R3B(OH)2,
Pd(PPh3)4,
15; hydrazine
O
R3
= 16; phenyl
17; 4-fluorophenyl
18; 2-furan
N
N
N
19; 2-thienyl
N
H
N
20; 4-chlorophenyl
R3
N
N
21; 4-trifluoromethylphenyl
H
16-21
Scheme 1. Synthesis of triazine-benzmidazoles
Table 1. Effects of 10 ꢀM of compounds 6, 16, 17 and 20 on cell proliferation in a subpanel
tumor cell lines.
Subpanel tumor cell lines
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
6
16
17
20
8.74
-22.74
-6.74
13.72
2.47
-15.74
-32.69
-30.14
-37.78
-22.64
8.94
-1.84
-46.01
-22.02
1.78
-13.83
6.01
-91.49
-81.44
-100.0
-91.61
-73.43
-83.32
9.79
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
13.89
27.70
-45.21
43.35
32.70
19.72
35.03
0.81
-68.45
-66.92
-64.49
-66.25
-19.29
-59.05
-0.47
11.23
7.91
-27.31
6.57
17.11
11.34
27.03
0.13
-75.19
-91.35
-73.89
-72.10
-64.52
-83.39
-92.85
-80.31
-73.93
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
SF-295
SF-539
SNB-19
SNB-75
U251
-67.25
-68.19
9.25
11.94
-61.28
-1.07
-51.43
7.95
10.38
-0.68
8.02
-68.80
-80.71
-76.36
-76.83
-18.63
-89.65
-67.51
-43.73
-19.85
-63.50
0.31
17.47
-19.21
6.38
-68.39
-89.86
-94.37
-92.73
-81.19
-92.64
-93.05
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
-77.07
-50.20
-24.53
-27.26
-20.55
-30.22
-90.75
-88.99
-77.26
-80.66
-81.06
-52.66
-82.85
-95.41
-75.60
-65.73
-7.64
-38.01
-19.20
-34.04
-90.65
-92.98
-73.43
-82.62
-86.10
-71.35
-84.68
-97.59

ACCEPTED MANUSCRIPT
UACC-257
Ovarian cancer
IGORV1
-19.30
-56.40
-27.63
-82.52
51.34
10.91
61.19
73.94
45.70
45.64
22.49
-38.08
-73.44
-47.23
0.63
-50.44
-60.81
-30.32
33.68
5.15
-71.16
-95.58
-73.91
-74.72
-73.82
-87.53
-89.89
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR- RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
59.84
66.34
39.24
34.23
19.61
-57.34
-28.59
22.92
-46.89
-56.14
52.41
58.80
-37.82
-88.93
-83.40
-58.90
-89.38
-75.14
-62.79
-58.06
-60.57
-60.02
-32.12
9.00
-56.60
-67.46
40.78
43.87
-43.52
-94.51
-81.51
-90.60
-93.57
-91.12
-79.48
-94.44
-87.59
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
12.41
38.96
-35.46
-27.79
2.31
38.47
-93.48
-93.96
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
3.49
23.15
-12.59
23.12
23.39
-3.66
-76.15
-63.34
-39.90
33.65
-62.31
-62.73
-44.29
5.74
-11.86
5.16
16.08
-1.23
-73.81
-76.74
-40.25
-73.63
-64.77
-83.99
MDA-MB-468
20-40% growth inhibition; 40-50% growth inhibition; 50-70% growth inhibition; 70-80% growth
inhibition; 80-90% growth inhibition; 90-100% growth inhibition; highly potent compounds.
Table 2. Compounds 6, 16, 17 and 20 with median growth inhibitory (GI50, µM), total
growth inhibitory (TGI, µM) and median lethal concentrations (LC50, µM) of in vitro tumor
cell lines at five different concentrations viz., 10-4, 10-5, 10-6, 10-7 and 10-8 M
Compds.
Activity
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
I
II
III
IV
V
VI
VII
VIII
3.67
IX
MIG-MIDa
2.68
2.37
2.85
9.66
9.54
1.74
3.67
14.2
2.68
6.65
7.76
1.15
2.59
5.58
2.43
5.52
30.2
0.99
2.55
6.53
2.15
4.73
7.90
0.26
0.69
2.75
2.54
23.2
46.6
1.41
3.04
5.98
2.23
11.8
8.47
0.77
1.95
4.18
11.8
1.56
3.18
6.52
1.91
4.04
7.43
1.07
2.48
5.79
3.46
22.6
26.9
1.85
3.88
6.84
2.74
2.46
11.8
8.29
1.20
2.72
6.27
2.03
7.36
7.83
1.11
2.25
5.60
2.36
6.72
92.7
1.63
4.08
8.74
2.09
6
6.50
9.13
11.0
32.3
1.38
3.15
8.63
2.37
7.16
7.88
b
b
0.57
2.17
16.3
2.02
1.51
3.07
6.25
3.46
16
17
20
6.11
11.5
6.66
5.55
b
b
b
b
0.19
0.92
2.30
5.20
0.41
1.49
4.33
0.62
0.72
1.80
4.88
0.58
1.92
1.86
b
b
b
LC50
I, leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer;
VII, renal cancer; VIII, prostate cancer; IX, breast cancer. a Full panel mean-graph midpoint (ꢀM). b Compounds
showed values >100 ꢀM.

ACCEPTED MANUSCRIPT
Figure 2. Absorption spectral changes of BSA (10 ꢀM) in phosphate buffer upon addition of
compounds 6 (a), 16 (b), 17 (c) and 20 (d) at 0-20 ꢀM; Insets indicate Benesi–Hildebrand plot
of 1/[A0-A] vs. 1/[compounds] for binding of BSA with compounds 6 (a), 16 (b), 17 (c) and
20 (d) through absorption spectral titrations.
Figure 3. Fluorescence spectral changes of BSA (10 ꢀM) in phosphate buffer upon addition
of 6 (a), 16 (b), 17 (c) and 20 (d) at 0-20 ꢀM. Insets indicate Benesi–Hildebrand plot of 1/[F0-
F] vs. 1/[compounds] for binding of BSA with compounds 6 (a), 16 (b), 17 (c) and 20 (d)
through emission spectral titrations.

ACCEPTED MANUSCRIPT
Table 3. Photophysical properties and binding constants of BSA with compounds 5-14, 16,
17 and 20.
Sr.
No.
Hyperchromicity
(%) in absorption (%) in emission
Enhancement
Isoemissive
point
Red shift in
emission
intensity (nm)
24
K (absorption K (emission
spectra, M-1) spectra, M-1)
n
spectra
72.93
spectra
63.43
(nm)
350
7.0 × 104
9.7 × 104
1.0 × 105
8.5 × 104
8.4 × 104
1.2 × 105
6.3 × 104
9.5 × 104
1.2 × 104
1.0 × 105
1.2 × 105
1.0 × 105
1.0 × 105
6.9 × 104
5.7 × 104
5.4 × 104
2.6 × 104
5.7 × 104
5.4 × 104
1.3 × 105
8.0 × 104
1.8 × 105
1.6 × 105
3.2 × 104
7.9 × 104
4.2 × 107
1.24
0.91
1.67
1.19
0.90
1.61
0.94
1.01
1.14
1.58
1.47
0.99
0.87
5
72.31
65.29
75.98
85.95
68.72
70.53
90.60
88.44
85.87
72.27
86.80
97.67
49.74
68.99
77.53
57.22
73.21
69.01
71.82
42.06
23.12
22.78
71.83
49.10
350
341
339
345
343
347
340
352
349
357
341
349
25
22
22
23
25
22
23
24
17
20
23
22
6
7
8
9
10
11
12
13
14
16
17
20
Figure 4. Overlap of the fluorescence emission spectra of BSA with UV-Visible absorption
spectra of compounds 6 (a), 16 (b), 17 (c) and 20 (d).

ACCEPTED MANUSCRIPT
Table 4. Forster energy transfer parameters for BSA and triazine-benzimidazoles (5-14, 16,
17 and 20).
Sr. No.
Φ (ligand)
Φ (complex) % Overlapping
Energy
efficiency (E)
37.50
J (mol-1 cm-1 nm4 ) R0 (nm) r (nm)
area
0.31
0.24
0.23
0.30
0.36
0.34
0.23
0.32
0.38
0.28
0.22
0.31
0.30
0.20
0.29
0.23
0.25
0.24
0.22
0.27
0.22
0.29
0.28
0.29
0.20
0.31
24.98
30.29
9.60
1.25 × 1015
1.28 × 1015
1.35 × 1015
1.21 × 1015
1.29 × 1015
1.28 × 1015
1.24 × 1015
1.46 × 1015
1.42 × 1015
1.44 × 1015
1.59 × 1015
1.39 × 1015
1.63 × 1015
4.41
4.43
4.47
4.40
4.44
4.43
4.41
4.53
4.51
4.52
4.59
4.49
4.61
4.78
7.90
5.28
5.45
5.38
5.14
5.81
5.13
6.94
6.40
6.61
7.62
7.98
5
2.63
26.86
21.62
24.41
29.30
15.58
31.71
7.39
6
7
2.30
8
14.50
19.35
19.46
10.78
26.74
18.57
27.55
23.80
37.63
9
10
11
12
13
14
16
17
20
11.28
9.56
3.74
3.10

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
A series of triazine-benzimidazole analogs has been synthesized.
Compounds were evaluated in vitro for their antitumor activity.
Four compounds showed excellent activity towards 60 human cancer cell lines.
Interaction with bovine serum albumin has been determined.