Triazine-benzimidazole hybrids: Anticancer activity, DNA interaction and dihydrofolate reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.03.012

A new series of triazine-benzimidazole hybrids has been synthesized with different substitution of primary and secondary amines at one of the position of triazine in moderate to good yields. These compounds were evaluated for their inhibitory activities over 60 human tumor cell lines at one dose and five dose concentrations. Compounds 6b, 8 and 9 showed broad spectrum of antitumor activities with GI₅₀ values of 9.79, 2.58 and 3.81 μM, respectively. DNA binding studies also indicated strong interaction properties of these compounds. These synthesized compounds also showed inhibition of mammalian dihydrofolate reductase (DHFR). Compound 6b was depicted as the most active member of DHFR inhibitor with IC₅₀ value of 1.05 μM. Molecular modelling studies were used to identify the stabilized interactions of Compound 6b within the active site of enzyme for DHFR.

Full text of DOI:10.1016/j.bmc.2015.03.012

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Triazine–benzimidazole hybrids: Anticancer activity, DNA interaction
and dihydrofolate reductase inhibitors
⇑
Prinka Singla, Vijay Luxami, Kamaldeep Paul
School of Chemistry and Biochemistry, Thapar University, Patiala 147001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of triazine–benzimidazole hybrids has been synthesized with different substitution of pri-
mary and secondary amines at one of the position of triazine in moderate to good yields. These com-
pounds were evaluated for their inhibitory activities over 60 human tumor cell lines at one dose and
five dose concentrations. Compounds 6b, 8 and 9 showed broad spectrum of antitumor activities with
Received 7 January 2015
Revised 2 March 2015
Accepted 3 March 2015
Available online xxxx
GI50 values of 9.79, 2.58 and 3.81 lM, respectively. DNA binding studies also indicated strong interaction
properties of these compounds. These synthesized compounds also showed inhibition of mammalian
dihydrofolate reductase (DHFR). Compound 6b was depicted as the most active member of DHFR inhibi-
Keywords:
Triazine–benzimidazole hybrids
Anticancer
DHFR
DNA interaction
Molecular modelling
tor with IC50 value of 1.05
tions of Compound 6b within the active site of enzyme for DHFR.
Ó 2015 Elsevier Ltd. All rights reserved.
lM. Molecular modelling studies were used to identify the stabilized interac-
1. Introduction
Topoisomerase 1,11 DNA and RNA polymerases 1.12 Some highly
efficacious triazine hybrids (A–C)13,14 are exemplified in Figure 1.
Encouraged by the activity profile of triazine and benzimida-
zole, and as a part of our ongoing research towards the design
and development of novel anticancer entities, we have combined
the structural artifacts of s-triazine and benzimidazole moieties
as hybrid that is enriched with electronic environment to get single
molecular framework. These hybrid molecules consisted of a
The molecular hybridization is a strategy of rational design of
new chemical entities based on the recognition of pharmacophoric
sub-units in the molecular structure of two or more known bioac-
tive derivatives. The adequate fusion of these sub-units, lead to the
design of new hybrid architectures that maintain pre-selected
characteristics of the original templates.1 It is a new concept in
drug design and development to produce a new hybrid compounds
with improved affinity and efficacy, when compared to the parent
drugs. The selection of the two chemical entities in the drugs is
usually based on their observed synergistic or additive pharmaco-
logical activities to enable the identification of highly active novel
chemical entities to counter balance the known side effects associ-
ated with the other hybrid part. Owing to immense synthetic
importance and varied bioactivities of triazine derivatives viz.,
anticancer,2 antimalarial,3 antimicrobial,4 antiviral, antiprotozoal,
and antileshmanial5 several attempts were made for modifying
the triazine nucleus to improve their activities. Similarly,
benzimidazoles6 have also endowed their selective binding to
parasite b-tubulin,7 specifically to DNA minor groove, serotonin
receptors (5-HT), histamine (H4) receptors, dopamine (D4) recep-
OCH3
HN
MeO
N
N
O
Cl
N
N
N
N
H
N
O
N
H
N
N
N
N
N
N
O
B
A
S
NR1R2
N
tors,8 VEGFR-2 tyrosine kinase receptors,9
G protein-coupled
N
N
N
receptors (GPCRs), thymidylate kinase, NK2 antagonist,10 a large
array of DNA and RNA processing enzymes such as
N
N
NH
CH3
N
N
N
H
N
N
O
7-24
OH
C
⇑
Corresponding author. Tel.: +91 9465670595; fax: +91 175 236 4498.
E-mail address: kpaul@thapar.edu (K. Paul).
Figure 1. Triazine (A–C) and proposed triazine–benzimidazole (7–24) hybrids.
http://dx.doi.org/10.1016/j.bmc.2015.03.012
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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2
P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
H2N
N
N
Benzyl chloride, K2CO3,
O2N
CH3
N
N
C2H5OH, 80 o
C
SnCl2.2H2O
CH3
2N HCl, 110 o
C
C
4
7
3
NH2
NH2
O2N
H2SO4
N
CH3COOH
N
5
CH3
CH3
100 o
C
N
H
2
1
4a
6
HNO3, 0 o
C
N
H
3a
N
N
2
1
N
SnCl2.2H2O
CH3
CH3
H2N
N
O2N
Benzyl chloride, K2CO3,
C2H5OH, 80 o
2N HCl, 110 o
C
4b
3b
Scheme 1. Synthesis of substituted aminobenzimidazoles.
Cl
N
Cl
N
Cl
N
Cl
N
Cl
N
N
N
N
N
N
4b
N
morpholine
morpholine
4a
N
N
N
N
N
N
CH3
N
N
N
CH3
CH3
CH3
N
N
H
NaHCO3
,
N
N
NaHCO3
THF, rt
,
N
N
H
NaHCO3
,
Cl
N
H
NaHCO3,
THF, rt
Cl
Cl
Cl
N
N
H
O
THF, 0-5 °C
O
THF, 0-5 °C
6b
5a
6a
5b
Scheme 2. Synthesis of monosubstituted and disubstituted triazines.
NR1R2 = 7; morpholine
NR1R2
8;
9;
4-fluoroaniline
piperidine
Cl
N
N
HNR1R2, K2CO3
,
N
R3
N
CH3
R3B(OH)2, Pd(PPh3)4,
N
N
10; N-methylpiperazine
11; pyrrolidine
12; 1,2-ethylenediamine
13; 1,2-ethanolamine
N
N
N
N
N
N
CH3
N
N
N
H
N
N
1,4-dioxane, 110 0
C
CH3
N
N
N
N
H
O
K2CO3, 1,4-dioxane,
reflux
N
N
N
H
O
O
7-14
O
6a
22-24
14; 2-aminoethylmorpholine
Cl
N
R3
22;
23;
24;
NR1R2
N
=
phenyl
NR1R2
=
; morpholine
; piperidine
; pyrrolidine
; N-methylpiperazine
; 1,2-ethylenediamine
; 1,2-ethanolamine
; 2-aminoethylmorpholine
15
16
17
18
19
20
21
N
N
4-fluorophenyl
4-chlorophenyl
HNR1R2, K2CO3
,
N
N
CH3
CH3
N
H
N
N
N
N
1,4-dioxane, 110 0C
H
O
6b
15-21
Scheme 3. Synthesis of 2,4,6-trisubstituted triazines from compounds 6a and 6b.
heterocyclic ring (triazine) as central core that can act as a scaffold
to carry three functionalized branches at 2-, 4- and 6-positions. The
regioisomeric aminobenzimidazole ring has been accommodated
at 2-position, morpholine at 4-position and different primary as
well as secondary amines at 6-position of triazine in order to
observe the effect of donor and acceptor nitrogen groups within
the moieties. Introduction of different orientations of benzyl group
at 1- or 3-position of regioisomeric aminobenzimidazole moiety
can also affect the activity of the hybrids. These compounds were
screened for their in vitro antitumor activities in full NCI 60 cell
lines panel assay and DNA interactions with different spectroscopy
techniques as well as DHFR immunoassay.
2. Chemistry
Figure 2. 2D NOE’s proton–proton correlations used for structural assignment of
compounds 4a and 4b.
The synthesis of triazine–benzimidazole hybrids and their sub-
stitution with primary and secondary amines as well as Suzuki
couplings (7–24) has been depicted in Schemes 1–3. The
aminobenzimidazoles 4a and 4b were prepared by benzylation of
2-methyl-5-nitro-1H-benzimidazole (2) with benzyl chloride in
the presence of potassium carbonate at 80 °C to give mixture of
3a and 3b followed by reduction with stannous chloride in 2 N
HCl at 110 °C (Scheme 1). Discrimination between N-substituted
products 4a and 4b was achieved by considering 2D NOE difference
experiments. Positive NOE’s on signals of N-CH2 protons due to the
benzyl chain and doublet of phenyl ring of benzimidazole revealed
compound 4a and positive NOE’s on signal of N-CH2 protons due to
the benzyl chain and singlet of aromatic ring of benzimidazole and
negative NOE with doublet, confirmed the formation of compound
4b (Fig. 2). Compound 4a was further treated with cyanuric chlo-
ride in the presence of 10% sodium bicarbonate and THF at 0–
5 °C for 6 h to give (1-benzyl-2-methyl-1H-benzimidazol-5-yl)-
(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (5a) in 96% yield followed
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P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Table 1
Compounds 6b, 8, 9, triazine and benzimidazole analogue median growth inhibitory (GI50
,
l
M), total growth inhibitory (TGI,
l
M) and median lethal concentrations (LC50
,
l
M) of
in vitro subpanel tumor cell lines
Compds
Activity
I
II
III
IV
V
VI
VII
VIII
IX
MG MIDa
6b
GI50
TGI
LC50
18.6
b
b
11.9
17.4
7.89
3.31
35.6
7.22
10.3
53.0
b
3.81
16.2
6.18
3.56
21.2
b
12.1
40.0
45.7
19.0
b
b
5.61
15.3
67.9
9.79
28.3
35.2
8
GI50
TGI
LC50
3.71
5.49
b
2.56
4.02
b
2.76
3.65
b
1.86
b
b
2.70
4.18
b
2.41
3.98
b
1.89
3.18
5.88
2.75
b
b
2.58
5.47
b
2.58
4.28
5.88
9
GI50
TGI
LC50
2.50
7.65
b
5.57
22.1
75.6
2.61
10.2
19.6
3.73
19.5
38.7
3.15
10.7
46.9
5.98
25.3
58.6
2.96
13.5
30.9
3.86
17.8
61.2
4.00
23.3
59.1
3.81
16.6
48.8
Triazine analogue
GI50
TGI
LC50
1.18
2.92
3.46
3.84
11.5
27.1
4.47
14.6
44.3
7.05
18.9
44.2
20.42
14.0
35.5
6.82
19.5
48.0
1.46
12.0
36.3
3.31
4.63
13.0
1.48
22.4
49.4
5.49
15.4
42.6
Benzimidazole
analogue
GI50
TGI
LC50
17.4
b
b
62.0
38.2
b
6.32
b
b
6.80
34.8
51.5
17.5
b
b
21.8
b
b
11.2
36.8
b
7.90
23.8
61.8
12.1
b
b
18.1
33.4
56.7
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 (
l
M.
M).
b
Compounds showed values >100
l
by stirring with morpholine at room temperature in the presence
of 10% sodium bicarbonate and THF for 4 h gave compound 6a in
80% yield (Scheme 2).
respectively, and more specific towards colon, melanoma, ovarian
and breast cancer cell lines with GI50 values in the range of 3.31–
5.61 lM. Nucleophilic displacement with primary and secondary
Similarly, compound 4b was treated with cyanuric chloride in
the same reaction conditions to obtain (3-benzyl-2-methyl-3H-
benzimidazol-5-yl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (5b)
in 90% yield followed by nucleophilic substitution with morpholine
to obtain compound 6b in 74% yield. Alternatively, poor overall
yields of 6a–b were obtained with first nucleophilic substitution
of cyanuric chloride with morpholine followed by substitution
with aminobenzimidazoles (4). Finally, treatment of compounds
6a–b with different primary as well as secondary amines in the
presence of potassium carbonate in 1,4-dioxane at 110 °C for 8 h
gave compounds 7–21 in moderate to good yields. Compound 6a
was also treated with different boronic acids viz., phenyl, 4-fluoro-
phenyl and 4-chlorophenyl boronic acids through Suzuki reaction,
in the presence of Pd(PPh3)4 and K2CO3 in 1,4-dioxane afforded
compounds 22–24 in 69–78% yields (Scheme 3). The structures
of newly synthesized compounds were confirmed by 1H and 13C
NMR as well as mass spectrometry (Supporting information).
amines enhanced the antitumor activity by 2–4 folds. Compound
8 exhibited remarkable anticancer activity against CNS cancer
and renal cancer cell lines with GI50 values of 1.86 and 1.89 lM,
respectively. Similarly, compound 9 showed potency against leu-
kemia, colon and renal cancer cell lines with GI50 values ranging
from 2.50–2.96 lM. Anticancer activities of triazine–benzimida-
zole hybrids (6b, 8–9) were also compared with reported triazine
and benzimidazole derivatives. Compound 8 showed almost two
and seven fold more activity than respective 2-amino-6-bro-
momethyl-4-(3,5,5-trimethyl-2-pyrazoline)-1,3,5-triazine19 and
benzimidazole (6-amino-5-(5,6-dimethyl-1H-benzimidazol-2-yl)-
4-(4-methoxy-phenyl)pyridine-3-carbonitrile) analogue,20 with
full panel mean graph mid-point (MG MID) GI50, TGI and LC50 val-
ues of 2.58, 4.28 and 5.88 lM, respectively. Compound 9 also
showed almost two and five fold more activity than respective tri-
azine and benzimidazole derivative, with MG MID GI50, TGI and
LC50 values of 3.81, 16.6 and 48.8 lM, respectively.
Due to the presence of the aromatic ring systems and protonat-
ed amine sites in triazine–benzimidazole hybrids,21,22 DNA inter-
action or enzyme inhibition may participate for the tumor
growth inhibitory activities of compounds 6b, 8 and 9. This led
us to check the interaction of these compounds with calf thy-
mus-DNA (ct-DNA) (responsible for polymerase for protein synthe-
sis) and DHFR (enzymes involved in the process of propagation of
cancer). For this purpose, UV–Visible and fluorescence studies
along with thermal denaturation experiments have been per-
formed for ct-DNA as well as enzyme immunoassay with DHFR.
3. In vitro evaluation for antitumor activity
All the synthesized compounds were submitted to National
Cancer Institute (NCI) disease-oriented human cell lines for their
evaluation of in vitro antitumor activities.15–18 Thirteen com-
pounds (6b, 7–10, 12, 15, 17–21, 24) were evaluated against 60 cell
lines at one dose concentration, that is, 10ꢀ5 M which included
nine tumor subpanels namely; leukemia, non-small lung, colon,
CNS, melanoma, ovarian, renal, prostate and breast cancer cells
and their outputs were reported as a mean graph of the percent
growth of treated cells, and presented as percentage growth inhi-
bition (GI%) (Tables S1 and S2). Compounds 6b, 8 and 9 were fur-
ther screened against a panel of 60 different tumor cell lines at
4. DNA interaction studies
DNA binding studies have been considered as one of the possi-
ble mechanism for anticancer activity using spectrophotometry,
spectrofluorimetry, DNA melting etc.23 The mechanism of many
anticancer drugs relies upon the interaction with ct-DNA presum-
ably through ionic interactions between phosphate groups of DNA
and protonated amine sites in addition to the interactions between
aromatic nuclei of the drug with nucleotide bases, resulting in sta-
bilization of the helical configuration of DNA against thermal
denaturation.24,25 We therefore investigated the binding behavior
of compounds 6b, 8 and 9 (studied at 5-dose concentration)
5-dose concentration range viz., 10ꢀ4 10ꢀ5 10ꢀ6 10ꢀ7 and
, , ,
10ꢀ8 M as these have shown prominent cell growth inhibition at
10ꢀ5 M concentration against variety of cancer cell lines (Table 1,
Figs. S71–S79).
Three response parameters; median growth inhibitory (GI50
M), total growth inhibitory (TGI, M) and median lethal concen-
trations (LC50 M) were monitoring for each cell line (Table 1). MG
MID GI50, TGI and LC50 values (over all the 60 cancer cell lines) of
compound 6b has been shown to be 9.79, 28.3 and 35.2 M,
,
l
l
,
l
l
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4
P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
towards ct-DNA through stepwise addition of DNA to a solution of
the compounds at constant concentration and physiological pH.
Compound 8 (25 lM) showed absorption maxima at 255 nm and
Binding constant of compounds 6b, 8 and 9 with ct-DNA
Compds
Ka (UV studies) (Mꢀ1
)
Ka (fluorescence studies) (Mꢀ1
)
306 nm (Fig. 3a). Upon incremental addition of ct-DNA to the solu-
tion of compound 8, there is increase in the absorption intensity at
kmax 255 nm with concomitant decrease in the absorption intensity
at kmax 306 nm with isobestic point at 294 nm. The presence of iso-
bestic point during titration with compound 8 and DNA revealed
that different species are in equilibrium and there is no stepwise
formation of the complexed species. In case of emission spectra,
compound 8 caused emission quenching in the presence of DNA
(Fig. 3b).
6b
8
9
1.01 ꢁ 105
3.06 ꢁ 104
3.75 ꢁ 104
7.84 ꢁ 104
5.46 ꢁ 104
2.6 ꢁ 104
interaction with nucleic acids. In order to evaluate strength of
intercalative nature of compounds with DNA, the thermal behavior
of ct-DNA in the presence of hybrids was also determined
(Table S3).
Intercalation of molecules into the double helix is known to sta-
bilize the DNA against thermal strand separation and thus increas-
es thermal melting temperature (Tm). The derivative melting
curves showed an increase of 2.6 °C and 15 °C in thermal melting
temperatures as compared to ct-DNA (60 °C) upon addition of
compounds 8 (62.6 °C) and 9 (85.0 °C), respectively. Thus, UV–
Visible and fluorescence titrations as well as thermal denaturation
experiments indicated strong intercalative nature of compounds
6b, 8 and 9 with ct-DNA.
The stepwise increase in concentrations of DNA in case of com-
pound 6b caused increase in the absorption intensity along with
tailing in absorption intensity between 325 and 1000 nm which
indicated the possibility of aggregation of compound in the pres-
ence of DNA whereas compound 9 caused decrease in absorption
intensity at their respective kmax (Figs. S80–S81). In case of emis-
sion spectra, compound 6b caused fluorescence enhancement
typically due to aggregation whereas compound 9 showed emis-
sion quenching in the presence of DNA. The binding constant Ka
for ligand–DNA binding was determined from the Benesi–
Hildebrand26 equation for UV–Vis and fluorescence titrations, sug-
gested strong interaction of ct-DNA with compound 8 that showed
5. Dihydrofolate reductase inhibition
Dihydrofolate reductase (DHFR) inhibition assay kit, involving
the DHFR mediated conversion of dihydrofolate to tetrahydrofolate
in the presence of NADPH has been used to investigate the inhibi-
tion of DHFR in the presence of different concentrations of com-
pounds 6b, 8 and 9 spanning 5 orders of magnitude. It has been
observed that compounds 6b and 9 showed excellent inhibitory
Ka = 3.06 ꢁ 104 Mꢀ1
through
absorption
titration
and
5.46 ꢁ 104 Mꢀ1 through emission titration (Table 2). These binding
of ligand to nucleic acids of DNA may possibly induced conforma-
tional changes depending upon the strength and mode of its
activity with IC50 values of 1.05 and 2.07 lM, respectively
(Table 3). However, compound 8 did not show any activity towards
DHFR enzyme immunoassay.
6. Structure–activity relationship studies
The structure–activity relationship emerged from the activity
towards cancer cell lines, DNA and DHFR revealed that permuta-
tion of triazine and benzimidazole hybrid was well tolerated for
in vitro anticancer and DHFR inhibitory activity. With these stud-
ies, it has been observed that compounds 6a and 7–14 showed
comparatively higher activity than their isomer 6b and 15–21 sug-
gested that there is much difference in antitumor activity with ori-
entation of benzyl group of benzimidazole. Regarding 60 human
tumor cell lines, in the series of compounds 6a and 7–14 substitu-
tion at C6 position of triazine with primary amines, 4-fluoroaniline
(8) showed higher activity than secondary amine, piperidine (9).
While in another series of compounds (6b and 15–21), chloro sub-
stitution at C6 position of triazine showed selectivity as compared
to the substitution of primary and secondary amines. In case of
DHFR assay study, chloro substitution at C6 position of triazine
(6b) showed excellent inhibitory activity.
7. Molecular modelling studies
In order to have an insight into the molecular interaction of
compounds with DHFR, their docking studies were planned to
Table 3
50% inhibitory concentration (IC50) of compounds 6b, 8 and 9 for
DHFR
Compds
IC50 (lM)
6b
8
9
1.05
NAa
2.07
Figure 3. Effect of addition of ct-DNA (3–30
lL) on (a) absorption (b) emission
a
Not active.
spectra of compound 8 (25 M) in phosphate buffer (pH 7.0).
l
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P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
8. Conclusions
The present work led to the development of novel antitumor
molecules containing regioisomeric hybrids of triazine and
benzimidazole moieties with substitution of primary and sec-
ondary amines. Compounds 6b, 8 and 9 with MG MID GI50 values
of 9.79, 2.58 and 3.81 lM, respectively, proved most active mem-
bers in 60 human cancer cell lines. These compounds also showed
strong interaction with DNA through UV–Visible and fluorescence
spectroscopy as well as thermal denaturation experiments. As per
the results of enzyme immunoassay, compounds 6b and 9 also
exhibited significant inhibition of DHFR with IC50 values of 1.05
and 2.09 lM, respectively. Moreover, molecular docking analysis
of most active compound 6b in the active site of DHFR also sup-
ported the experimental results for their interactions. Overall
results revealed that: (i) regioisomeric aminobenzimidazoles at tri-
azine ring play a key role in varying the efficiency of antitumor
activities, (ii) morpholine at one of position of triazine ring
positively influences the antitumor effectiveness, inducing good
activity against most of the cancer cell lines, (iii) chloro substitu-
tion at C6-position of triazine ring also imparts antitumor activity,
(iv) introduction of 4-fluoroaniline (8) and piperidine (9) at the C6-
position of triazine ring led to a broad spectrum of selectivity with
respect to other primary and secondary amines, thus essential for
activity, (v) linker between triazine and benzimidazole is essential
as H-bond region.
9. Experimental section
9.1. Chemistry
All materials were obtained from commercial suppliers and
used without further purification unless otherwise noted.
Anhydrous THF was distilled from sodium. Reactions were moni-
tored by thin-layer chromatography (TLC) carried out on silica
gel plates (GF 254) using UV light as visualizing agents. Silica gel
(60–120 mesh) was used for column chromatography. UV–Vis
studies were carried out on a Specord PC machines using slit width
of 1.0 nm and matched quartz cells. The fluorescence spectra were
determined on a Varian Cary Eclipse fluorescence spectrometer. 1H
NMR and 13C NMR spectra were recorded on a Bruker Avance 400
(1H, 400 MHz; 13C, 100 MHz) or Jeol-400 (1H, 400 MHz; 13C,
100 MHz) spectrometer at ambient temperature, using CDCl3 and
DMSO-d6 as solvents. Chemical shifts are reported in ppm. Data
are reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), cou-
pling constants and number of protons. Mass spectrometric data
were obtained using MAT 120. Hexane: ethyl acetate and chloro-
form: methanol was the adopted solvent systems.
Figure 4. (A) Compound 6b docked in the active site of DHFR (3GHW) (B)
Hydrophobic groups are shown in gray in colour and hydrogen bonded groups are
orange in colour. The p–p interactions are shown as black lines.
scrutinize the mode of interactions of compounds with amino
acids in the active site of the enzymes.27 The crystal coordinates
of DHFR complex (PDB ID 3GHW)28 were downloaded from protein
data bank (www.rcsb.org). Molecular modelling was carried out
with compound 6b which has been proved to be the most active
in DHFR inhibition assay. Compound 6b sits in the active site of
the enzyme interacting through H-bonding between O group of
morpholine (d = 1.17 Å) with L22 amino acid residue coil of
enzyme (Fig. 4).
9.1.1. General procedure for the preparation of 1/3-benzyl-2-
methyl-1H/3H-benzimidazol-5-ylamine (4a, 4b)
NDP residues also showed H-bonding interactions with NH
group of benzimidazole (d = 2.44 Å) with flexible ligand,
Ascore = 2.783. Of the 21 best poses, selected based on their scor-
ing function, the best pose was used for the binding energy calcu-
lations (best ligand pose energy = ꢀ6.0187 kcal/mol) and further
analysis. Compound 6b also showed hydrophobic interaction with
To a solution of 2-methyl-5-nitro-1H-benzimidazole (2) (9.0 g,
0.05 mol) in ethanol (50 mL), K2CO3 (6.98 g, 0.075 mol) and benzyl
chloride (9.63 g, 0.075 mol) were refluxed for 8 h. After the com-
pletion of the reaction (monitored by TLC), the reaction mixture
was filtered and concentrated to give yellow solid. The residual
mass was crystallized from ethanol to give mixture of 1-benzyl-
2-methyl-5-nitro-1H-benzimidazole and 3-benzyl-2-methyl-5-ni-
amino acids as shown in Figure 4B. The
formed between the benzimidazole and phenyl ring of benzyl
group with Phe34. Besides, interactions were also observed
p–p interactions are
p–p
tro-3H-benzimidazole (3) in 75% yield. To
a suspension of
between the triazine ring and Trp624. Additionally, the benzimida-
zole and triazine displayed van der Waal’s interaction with Ile10,
Asp21, Lys18, Arg70 and Leu22 of 3GHW. Therefore, the crystal
coordinates of the DHFR in association with compound 6b support-
ed the experimental results, as these compounds exhibited interac-
tions with active site of enzymes.
SnCl2ꢂ2H2O (15.45 g, 0.067 mol) in 2 N HCl (47.6 mL), 1/3-benzyl-
2-methyl-5-nitro-1H/3H-benzimidazole (3) (4.25 g, 0.019 mol)
was heated at 110 °C for 7 h. After completion of the reaction,
the suspension was neutralized with 2 N NaOH and diluted with
ethanol. Filtered the solid product and extracted the filtrate with
Please cite this article in press as: Singla, P.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.012

6
P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
chloroform, dried over Na2SO4, filtered and concentrated to get
mixture of products which were separated through column chro-
matography using ethyl acetate/methanol (19:1) as eluent to get
solid compounds 4a and 4b.
column chromatographed on silica gel in chloroform/methanol
(50:1) to afford the desired product (1-benzyl-2-methyl-1H-
benzimidazol-5-yl)-(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-
yl)-amine (6a) as white powder; (9.07 g, 80%); mp: >300 °C; 1H
NMR (400 MHz, CDCl3): d = 7.76 (br s, 1H, NH), 7.66 (d, 1H,
J = 8.72 Hz, ArH), 7.32–7.26 (m, 5H, ArH), 7.10 (d, 1H, J = 8.24 Hz,
ArH), 7.02 (s, 1H, ArH), 5.30 (s, 2H, N-CH2), 3.80 (s, 2H, mor-CH2),
3.67 (s, 2H, mor-CH2), 3.50 (s, 2H, mor-CH2), 3.40 (s, 2H, mor-
CH2), 2.55 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): d = 164.6,
163.8, 152.4, 139.4, 135.6, 132.8, 129.2, 128.1, 125.7, 119.2,
116.2, 102.1 (ArC), 66.7 (O-CH2), 66.5 (O-CH2), 46.9 (N-CH2), 44.0
(N-CH2), 14.0 (CH3); EIMS, m/z: 436.2 (M++1); Anal. Calcd for
9.1.1.1. 1-Benzyl-2-methyl-1H-benzimidazol-5-ylamine (4a).
Brownish powder; (2.6 g, 69%); mp: 137–139 °C; 1H NMR
(400 MHz, CDCl3): d = 7.48 (d, 1H, J = 8.24 Hz, ArH), 7.30–7.24 (m,
3H, ArH), 7.03 (d, 2H, J = 6.88 Hz, ArH), 6.63 (dd, 1H, 2J = 8.24,
3J = 1.84 Hz, ArH), 6.47 (d, 1H, J = 1.36 Hz, ArH), 5.19 (s, 2H, N-
CH2), 2.51 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): d = 149.2,
142.3, 136.7, 136.0, 129.0, 127.9, 127.8, 126.2, 119.5, 119.4,
112.0, 111.1, 95.1 (ArC), 47.0 (N-CH2), 13.9 (CH3); EIMS, m/z:
238.2 (M++1).
C22H22ClON7: C, 60.62; H, 5.09; N, 22.49, Found: C, 61.01; H,
5.41; N, 22.14.
9.1.1.2. 3-Benzyl-2-methyl-3H-benzimidazol-5-ylamine (4b).
Brownish powder; (1.0 g, 26%); mp: 137–139 °C; 1H NMR
(400 MHz, CDCl3): d = 7.28–7.24 (m, 3H, ArH), 7.02 (s, 1H, ArH),
7.01 (d, 2H, J = 5.52 Hz, ArH), 6.97 (d, 1H, J = 8.24 Hz, ArH), 6.60
(d, 1H, J = 8.24 Hz, ArH), 5.22 (s, 2H, N-CH2), 2.52 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3): d = 151.7, 143.2, 142.1, 135.9, 129.3,
129.0, 127.9, 126.2, 112.3, 109.8, 104.3 (ArC), 47.1 (N-CH2), 13.8
(CH3); EIMS, m/z: 238.2 (M++1).
9.1.5. (3-Benzyl-2-methyl-3H-benzimidazol-5-yl)-(4-chloro-6-
morpholin-4-yl-[1,3,5]triazin-2-yl)-amine (6b)
Intermediate 6b was prepared from 5b and morpholine accord-
ing to the method described for 6a as white powder; (8.38 g, 74%);
mp: >300 °C; 1H NMR (400 MHz, CDCl3): d = 8.08 (s, 1H, NH), 7.34–
7.30 (m, 3H, ArH), 7.23 (d, 1H, J = 3.68 Hz, ArH), 7.17 (d, 2H,
J = 8.72 Hz, ArH), 7.06 (d, 1H, J = 7.80 Hz, ArH), 7.04 (d, 1H,
J = 1.40 Hz, ArH), 5.32 (s, 2H, N-CH2), 3.85 (t, 4H, J = 9.16 Hz, mor-
CH2), 3.74–3.73 (m, 4H, mor-CH2), 2.58 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): d = 163.8, 152.9, 142.8, 135.6, 132.5, 129.3,
129.1, 128.1, 128.0, 126.2, 125.9, 122.9, 116.6, 116.5, 111.6, 109.4
(ArC), 66.7 (O-CH2), 47.3 (N-CH2), 44.1 (N-CH2), 14.1 (CH3); EIMS,
m/z: 436.2 (M++1); Anal. Calcd for C22H22ClON7: C, 60.62; H,
5.09; N, 22.49, Found: C, 60.65; H, 5.36; N, 22.30.
9.1.2. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-(4,6-dichloro-
[1,3,5]triazin-2-yl)-amine (5a)
To a stirred solution of cyanuric chloride (10.0 g, 0.054 mol) in
anhydrous THF (150 mL), 1-benzyl-2-methyl-1H-benzimidazol-5-
ylamine (4a) (11.56 g, 0.048 mol) in THF was added at 0–5 °C. To
this mixture, 10% NaHCO3 was added drop wise and stirred at
the same temperature for 6 h. The resulted reaction mixture was
then poured into crushed ice and filtered, dried and then column
chromatographed on silica gel in chloroform/methanol (50:1) as
eluents to give (1-benzyl-2-methyl-1H-benzimidazol-5-yl)-(4,6-
9.1.6. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-(4,6-di-mor-
pholin-4-yl-[1,3,5]triazin-2-yl)-amine (7)
To a stirred solution of (1-benzyl-2-methyl-1H-benzimidazol-
5-yl)-(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-amine (6a)
(0.200 g, 0.459 mmol) in 1,4-dioxane (20 mL), morpholine
(0.059 g, 0.688 mmol) and potassium carbonate (0.076 g,
0.551 mmol) were added and heated at 110 °C for 8 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 (1-benzyl-2-methyl-1H-benzimidazol-5-
yl)-(4,6-di-morpholin-4-yl-[1,3,5]triazin-2-yl)-amine (7) as white
powder; (0.158 g, 71%); mp: >300 °C; 1H NMR (400 MHz, CDCl3):
d = 7.94 (d, 1H, J = 1.80 Hz, ArH), 7.62 (d, 1H, J = 8.52 Hz, ArH),
7.32–7.26 (m, 3H, ArH), 7.07 (d, 3H, J = 1.92 Hz, ArH), 7.03 (s, 1H,
NH), 5.29 (s, 2H, N-CH2), 3.75–3.61 (m, 16H, mor-CH2), 2.51 (s,
3H, CH3); 13C NMR (100 MHz, CDCl3): d = 165.1, 164.2, 151.6,
138.1, 135.8, 134.8, 129.2, 128.0, 125.7, 118.9, 115.5, 101.1 (ArC),
66.8 (O-CH2), 46.7 (N-CH2), 43.6 (N-CH2), 13.9 (CH3); EIMS, m/z:
487.3 (M++1); Anal. Calcd for C26H30N8O2: C, 64.18; H, 6.21; N,
23.03, Found: C, 64.33; H, 6.29; N, 23.40.
dichloro-[1,3,5]triazin-2-yl)-amine (5a) as
a yellow powder;
(20.02 g, 96%); mp: >300 °C; 1H NMR (400 MHz, DMSO-d6):
d = 11.22 (s, 1H, NH), 7.98 (s, 1H, ArH), 7.60 (d, 1H, J = 3.60 Hz,
ArH), 7.42 (dd, 1H, 2J = 8.72 Hz, 3J = 1.92 Hz, ArH), 7.34–7.29 (m,
5H, ArH), 5.49 (s, 2H, N-CH2), 2.50 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6): d = 154.8, 152.2, 150.4, 134.3, 132.3, 129.5,
128.9, 128.5, 128.3, 120.6, 114.8, 105.3 (ArC), 48.3 (N-CH2), 12.4
(CH3); EIMS, m/z: 385.1 (M++1); Anal. Calcd for C18H14Cl2N6: C,
56.12; H, 3.66; N, 21.81, Found: C, 56.42; H, 3.60; N, 21.44.
9.1.3. (3-Benzyl-2-methyl-3H-benzimidazol-5-yl)-(4,6-dichloro-
[1,3,5]triazin-2-yl)-amine (5b)
Intermediate 5b was prepared from 4b and cyanuric chloride
according to the method described for 5a as yellow powder;
(18.78 g, 90%); mp: >300 °C; 1H NMR (400 MHz, DMSO-d6):
d = 8.60 (br s, 1H, NH), 8.07 (d, 1H, J = 1.84 Hz, ArH), 7.36 (d, 1H,
J = 8.72 Hz, ArH), 7.29–7.21 (m, 3H, ArH), 7.16 (d, 1H, J = 8.68 Hz,
ArH), 7.05 (d, 1H, J = 5.96 Hz, ArH), 6.85 (d, 1H, J = 8.68 Hz, ArH),
5.31 (s, 2H, CH2), 2.57 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-
d6): d = 154.8, 152.2, 150.4, 136.4, 134.2, 132.2, 129.5, 129.0,
128.3, 126.8, 120.7, 114.9, 105.0 (ArC), 48.4 (N-CH2), 12.0 (CH3);
EIMS, m/z: 385.1 (M++1); Anal. Calcd for C18H14Cl2N6: C, 56.12;
H, 3.66; N, 21.81, Found: C, 56.37; H, 3.41; N, 21.87.
9.1.7. N-(1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-N0-(4-fluoro-
phenyl)-6-morpholin-4-yl-[1,3,5]triazine-2,4-diamine (8)
By use of procedure of reaction 7, compound
8 using
4-fluoroaniline gave as white powder; (0.159 g, 68%); mp:
>300 °C; 1H NMR (400 MHz, CDCl3): d = 7.81 (br s, 1H, NH), 7.64
(d, 1H, J = 8.56 Hz, ArH), 7.47–7.32 (m, 2H, ArH), 7.31–7.26 (m,
3H, ArH), 7.13 (dd, 1H, 2J = 8.56 Hz, 3J = 1.84 Hz, ArH), 7.03–7.01
(m, 4H, ArH, NH), 6.98 (d, 1H, J = 8.6 Hz, ArH), 6.67 (s, 1H, ArH),
5.26 (s, 2H, N-CH2), 3.69 (m, 8H, mor-CH2), 2.53 (s, 3H, CH3); 13C
NMR (100 MHz, CDCl3): d = 165.1, 159.9, 151.5, 138.4, 135.8,
134.9, 134.0, 132.9, 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), 14.1
(CH3); EIMS, m/z: 511.3 (M++1); Anal. Calcd for C28H27FN8O: C,
65.87; H, 5.33; N, 21.95, Found: C, 65.37; H, 5.30; N, 21.77.
9.1.4. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-(4-chloro-6-
morpholin-4-yl-[1,3,5]triazin-2-yl)-amine (6a)
To
a
stirred solution of (1-benzyl-2-methyl-1H-benzi-
midazol-5-yl)-(4,6-dichloro-[1,3,5]triazin-2-yl)-amine (5a) (10 g,
0.026 mol) in anhydrous THF (150 mL), morpholine (2.69 g,
0.031 mol) was added at room temperature. To this mixture, 10%
NaHCO3 was added drop wise and stirred for 4 h. The resulted reac-
tion mixture was then poured into crushed ice, filtered, dried and
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P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
9.1.8. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-(4-
NMR (400 MHz, CDCl3): d = 8.07 (br s, 1H, NH), 7.61 (d, 2H,
J = 8.68 Hz, ArH), 7.32–7.26 (m, 3H, ArH), 7.07–7.05 (m, 1H, ArH),
6.85 (d, 2H, J = 8.28 Hz, ArH), 5.59 (br s, 1H, NH), 5.29 (s, 2H, N-
CH2), 3.69 (s, 6H, mor-CH2), 3.51 (s, 2H, mor-CH2), 2.56 (t, 2H,
J = 5.60 Hz, aliphatic-CH2), 2.50 (m, 2H, aliphatic-CH2), 2.44
(s, 3H, CH3), 2.04 (br s, 1H, OH); 13C NMR (100 MHz, CDCl3):
d = 166.8, 164.9, 151.8, 138.7, 135.9, 134.1, 129.1, 127.9, 125.9,
119.0, 116.2, 101.8 (ArC), 67.8 (O-CH2), 46.9 (N-CH2), 43.8 (N-
CH2), 43.6 (N-CH2), 14.0 (CH3); EIMS, m/z: 461.3 (M++1); Anal.
Calcd for C24H28N8O2: C, 62.59; H, 6.13; N, 24.33, Found: C,
62.70; H, 6.31; N, 24.65.
morpholin-4-yl-6-piperidin-1-yl)-[1,3,5]triazin-2-yl)-amine (9)
By use of procedure of reaction 7, compound 9 using piperidine
gave as white powder; (0.172 g, 77%); mp: >300 °C; 1H NMR
(400 MHz, CDCl3): d = 8.01 (s, 1H, ArH), 7.61 (d, 1H, J = 8.56 Hz,
ArH), 7.32–7.25 (m, 3H, ArH), 7.06 (d, 3H, J = 1.84 Hz, ArH), 6.78
(br s, 1H, NH), 5.28 (s, 2H, N-CH2), 3.75–3.61 (m, 12H, mor-CH2,
pip-CH2), 2.51 (s, 3H, CH3), 1.56 (t, 4H, J = 7.44 Hz, pip-CH2), 1.27
(t, 2H, J = 7.56 Hz, pip-CH2); 13C NMR (100 MHz, CDCl3):
d = 165.3, 164.8, 164.4, 151.5, 138.2, 135.9, 135.8, 135.1, 129.1,
127.9, 125.9, 118.9, 115.2, 100.8 (ArC), 66.7 (O-CH2), 46.8 (N-
CH2), 44.2 (N-CH2), 43.6 (N-CH2), 25.8 (CH2), 24.9 (CH2), 14.0
(CH3); EIMS, m/z: 485.3 (M++1); Anal. Calcd for C27H32ON8: C,
66.92; H, 6.66; N, 23.12, Found: C, 66.95; H, 6.48; N, 23.29.
9.1.13. N-(1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-6-mor-
pholin-4-yl-N0-(2-morpholin-4-yl-ethyl)-[1,3,5]triazine-2,4-
diamine (14)
9.1.9. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-[4-(4-methyl-
piperazin-1-yl)-6-morpholin-4-yl-[1,3,5]triazin-2-yl]-amine (10)
By use of procedure of reaction 7, compound 10 using
N-methylpiperazine gave as white powder; (0.176 g, 77%); mp:
>300 °C; 1H NMR (400 MHz, DMSO-d6): d = 9.12 (br s, 1H, NH),
8.51 (s, 1H, ArH), 8.02 (d, 1H, J = 7.20 Hz, ArH), 7.49 (dd, 1H,
2J = 29.84 Hz, 3J = 7.28 Hz, ArH), 7.25–7.18 (m, 3H, ArH), 6.97–
6.95 (m, 2H, ArH), 5.31 (s, 2H, N-CH2), 3.69–3.44 (m, 12H, mor-
CH2, piperazine-CH2), 2.51 (s, 3H, CH3), 2.45 (t, 4H, J = 4.36 Hz,
piperazine-CH2), 2.26 (s, 3H, N-CH3); 13C NMR (100 MHz, CDCl3):
d = 164.9, 164.8, 151.8, 138.6, 135.9, 135.7, 135.0, 134.2, 129.2,
129.1, 127.9, 126.0, 122.1, 119.0, 115.4, 115.2 (ArC), 54.7 (O-
CH2), 46.8 (N-CH2), 46.2 (N-CH2), 43.2 (N-CH2), 29.7 (N-CH3),
14.0 (CH3); EIMS, m/z: 500.3 (M++1); Anal. Calcd for C27H33N9O:
C, 64.91; H, 6.66; N, 25.23, Found: C, 64.65; H, 6.41; N, 25.34.
By use of procedure of reaction 7, compound 14 using
2-aminoethylmorpholine gave as white powder; (0.191 g, 83%);
mp: >300 °C; 1H NMR (400 MHz, CDCl3): d = 7.91 (br s, 1H, NH),
7.60 (d, 1H, J = 8.72 Hz, ArH), 7.30–7.27 (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),
5.41 (t, 1H, J = 6.40 Hz, NH), 5.28 (s, 2H, N-CH2), 3.67–3.47 (m,
20H, mor-CH2, aliphatic-CH2), 2.50 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): d = 165.2, 164.3, 151.7, 138.4, 135.9, 135.8,
134.7, 129.1, 127.9, 125.9, 118.9, 115.5, 101.4, 101.1 (ArC), 66.9
(O-CH2), 66.7 (O-CH2), 57.3 (N-CH2), 53.4 (N-CH2), 46.8 (N-CH2),
43.6 (N-CH2), 36.9 (N-CH2), 14.0 (CH3); EIMS, m/z: 530.3 (M++1);
Anal. Calcd for C28H35N9O2: C, 63.50; H, 6.66; N, 23.80, Found: C,
63.90; H, 6.32; N, 23.60.
9.1.14. (3-Benzyl-2-methyl-3H-benzimidazol-5-yl)-(4,6-di-mor-
pholin-4-yl-[1,3,5]triazin-2-yl)-amine (15)
9.1.10. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-(4-mor-
pholin-4-yl-6-pyrrolidin-1-yl-[1,3,5]triazin-2-yl)-amine (11)
By use of procedure of reaction 7, compound 11 using pyrro-
lidine gave as white powder; (0.159 g, 74%); mp: >300 °C; 1H
NMR (400 MHz, CDCl3): d = 8.11 (s, 1H, NH), 7.61 (d, 1H,
J = 8.52 Hz, ArH), 7.32–7.03 (m, 3H, ArH), 7.04 (dd, 2H,
2J = 8.36 Hz, 3J = 1.72 Hz, ArH), 7.02 (d, 1H, J = 9.28 Hz, ArH), 6.79
(s, 1H, ArH), 5.30 (s, 2H, N-CH2), 3.75–3.73 (m, 6H, mor-CH2),
3.72–3.69 (m, 4H, pyrr-CH2), 3.61–3.59 (m, 2H, mor-CH2), 2.52 (s,
3H, CH3), 1.95 (s, 4H, pyrr-CH2); 13C NMR (100 MHz, CDCl3):
d = 151.4, 151.3, 138.0, 135.9, 135.6, 135.2, 129.1, 127.8, 125.8,
118.9, 118.7, 115.1, 115.0, 100.8 (ArC), 66.8 (O-CH2), 46.8
(N-CH2), 46.0 (N-CH2), 43.6 (N-CH2), 29.7 (CH2), 25.4 (CH2), 14.0
(CH3); EIMS, m/z: 471.3 (M++1); Anal. Calcd for C26H30N8O: C,
66.36; H, 6.43; N, 23.81, Found: C, 66.37; H, 6.47; N, 23.76.
By use of procedure of reaction 7, compound 15 using morpho-
line gave as white powder; (0.158 g, 71%); mp: >300 °C; 1H NMR
(400 MHz, CDCl3): d = 8.04 (d, 1H, J = 1.80 Hz, ArH), 7.30–7.26 (m,
4H, ArH), 7.11 (d, 1H, J = 8.72 Hz, ArH), 7.05 (d, 3H, J = 7.80 Hz,
ArH, NH), 5.28 (s, 2H, N-CH2), 3.79 (s, 8H, mor-CH2), 3.72 (s, 8H,
mor-CH2), 2.57 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d = 165.3, 164.5, 152.4, 143.0, 135.9, 134.3, 131.6, 129.0, 128.0,
126.2, 116.2, 110.7, 109.1 (ArC), 67.0 (O-CH2), 66.9 (O-CH2), 47.1
(N-CH2), 47.0 (N-CH2), 43.7 (N-CH2), 14.1 (CH3); EIMS, m/z: 487.3
(M++1); Anal. Calcd for C26H30N8O2: C, 64.18; H, 6.21; N, 23.03,
Found: C, 64.32; H, 6.12; N, 22.69.
9.1.15. (3-Benzyl-2-methyl-3H-benzimidazol-5-yl)-(4-morpholin-
4-yl-6-piperidin-1-yl)-[1,3,5]triazin-2-yl)-amine (16)
By use of procedure of reaction 7, compound 16 using piperidine
gave as white powder; (0.172 g, 77%): mp: >300 °C; 1H NMR
(400 MHz, CDCl3): d = 8.03 (d, 1H, J = 1.84 Hz, ArH), 7.31–7.26 (m,
4H, ArH), 7.11 (d, 1H, J = 8.72 Hz, ArH), 7.04 (d, 2H, J = 6.88 Hz,
ArH), 6.90 (br s, 1H, NH), 5.28 (s, 2H, N-CH2), 3.79–3.71 (m, 12H,
mor-CH2, pip-CH2), 2.54 (s, 3H, CH3), 1.64 (d, 2H, J = 4.60 Hz, pip-
CH2), 1.57 (d, 4H, J = 5.12 Hz, pip-CH2); 13C NMR (100 MHz,
CDCl3): d = 165.5, 164.9, 164.6, 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), 47.1
(N-CH2), 44.2 (N-CH2), 43.7 (N-CH2), 31.0 (CH2), 25.9 (CH2), 25.0
(CH2), 14.1 (CH3); EIMS, m/z: 485.3 (M++1); Anal. Calcd for
9.1.11. N-(2-Amino-ethyl)-N0-(1-benzyl-2-methyl-1H-benzimi-
dazol-5-yl)-6-morpholin-4-yl-[1,3,5]triazine-2,4-diamine (12)
By use of procedure of reaction 7, compound 12 using 1,2-
ethylenediamine gave as white powder; (0.172 g, 82%); mp:
>300 °C; 1H NMR (400 MHz, CDCl3): d = 7.93 (d, 1H, J = 8.72 Hz,
ArH), 7.75 (br s, 1H, NH), 7.61 (d, 1H, J = 4.32 Hz, ArH), 7.28–7.26
(m, 3H, ArH), 7.08–6.99 (m, 3H, ArH), 5.25 (s, 2H, N-CH2), 3.67
(s, 12H, mor-CH2, aliphatic-CH2), 2.49 (s, 3H, CH3), 1.91 (br s, 2H,
NH2); 13C NMR (100 MHz, CDCl3): d = 165.1, 164.3, 151.8, 151.7,
135.8, 129.1, 127.9, 126.2, 126.0, 125.7, 119.0, 115.4, 101.0 (ArC),
66.8 (O-CH2), 46.9 (N-CH2), 46.7 (N-CH2), 43.6 (N-CH2), 29.7
(CH2), 14.0 (CH3); EIMS, m/z: 460.3 (M++1); Anal. Calcd for
C24H29N9O: C, 62.73; H, 6.36; N, 27.43, Found: C, 62.37; H, 6.41;
N, 27.63.
C27H32N8O: C, 66.92; H, 6.66; N, 23.12, Found: C, 66.68; H, 6.43; N,
23.52.
9.1.16. (3-Benzyl-2-methyl-3H-benzimidazol-5-yl)-(4-morpholin-
4-yl-6-pyrrolidin-1-yl-[1,3,5]triazin-2-yl)-amine (17)
By use of procedure of reaction 7, compound 17 using pyrro-
lidine gave as white powder; (0.159 g, 74%); mp: >300 °C; 1H
NMR (400 MHz, CDCl3): d = 8.15 (s, 1H, ArH), 7.30–7.26 (m, 4H,
ArH), 7.11 (d, 1H, J = 8.72 Hz, ArH), 7.05 (d, 2H, J = 7.76 Hz, ArH),
9.1.12. 2-[4-(1-Benzyl-2-methyl-1H-benzimidazol-5-ylamino)-
6-morpholin-4-yl-[1,3,5]triazin-2-ylamino]-ethanol (13)
By use of procedure of reaction 7, compound 13 using 1,2-etha-
nolamine gave as white powder; (0.173 g, 82%); mp: >300 °C; 1H
Please cite this article in press as: Singla, P.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.012

8
P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
6.83 (s, 1H, NH), 5.29 (s, 2H, N-CH2), 3.80 (t, 4H, J = 9.16 Hz, mor-
CH2), 3.73 (t, 4H, J = 9.20 Hz, mor-CH2), 3.60 (s, 2H, pyrr-CH2),
3.58 (s, 2H, pyrr-CH2), 2.55 (s, 3H, CH3), 1.92 (s, 4H, pyrr-CH2);
13C NMR (100 MHz, CDCl3): d = 165.1, 164.2, 163.7, 152.2, 143.0,
136.0, 134.8, 131.3, 129.0, 127.9, 126.2, 115.8, 110.3, 109.0 (ArC),
67.0 (O-CH2), 47.1 (N-CH2), 46.1 (N-CH2), 43.7 (N-CH2), 25.4
(CH2), 25.3 (CH2), 14.1 (CH3); EIMS, m/z: 471.3 (M++1); Anal.
Calcd for C26H30N8O: C, 66.36; H, 6.43; N, 23.81, Found: C, 66.37;
H, 6.49; N, 23.77.
1H, NH), 5.29 (s, 2H, N-CH2), 3.80 (s, 8H, mor-CH2), 3.71 (s, 8H,
mor-CH2), 3.50 (s, 2H, aliphatic-CH2), 2.53 (s, 3H, CH3), 2.45 (s,
2H, aliphatic-CH2); 13C NMR (100 MHz, CDCl3): d = 167.8, 167.2,
166.9, 152.4, 143.0, 135.9, 134.8, 132.9, 129.1, 128.0, 127.5,
126.2, 118.9, 109.1 (ArC), 69.1 (O-CH2), 57.4 (N-CH2), 53.4 (N-
CH2), 47.2 (N-CH2), 43.7 (CH2), 37.0 (CH2), 14.1 (CH3); EIMS, m/z:
530.2 (M++1); Anal. Calcd for C28H35N9O2: C, 63.50; H, 6.66; N,
23.80, Found: C, 63.55; H, 6.92; N, 23.88.
9.1.21. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-(4-morpholin-
4-yl-6-phenyl-[1,3,5]triazin-2-yl)-amine (22)
9.1.17. (3-Benzyl-2-methyl-3H-benzimidazol-5-yl)-[4-(4-methyl-
piperazin-1-yl)-6-morpholin-4-yl-[1,3,5]triazin-2-yl]-amine
(18)
To a stirred solution of (1-benzyl-2-methyl-1H-benzimidazol-
5-yl)-(4-chloro-6-morpholin-4-yl-[1,3,5]triazin-2-yl)-amine (6a)
(0.200 g, 0.435 mmol) in 1,4-dioxane (20 mL), phenylboronic acid
(0.0637 g, 0.516 mmol), Pd(PPh3)4 (10 mol%) and potassium car-
bonate (0.076 g, 0.516 mmol) were added and refluxed for 8 h in
an inert atmosphere. After 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 elu-
ents to give (1-benzyl-2-methyl-1H-benzimidazol-5-yl)-(4-mor-
pholin-4-yl-6-phenyl-[1,3,5]triazin-2-yl)-amine (22) as white
powder; (0.150 g, 69%); mp: >300 °C; 1H NMR (400 MHz, CDCl3):
d = 8.34 (d, 2H, J = 7.32 Hz, ArH), 8.02 (br s, 1H, NH), 7.67 (d, 1H,
J = 6.28 Hz, ArH), 7.48 (d, 1H, J = 7.32 Hz, ArH), 7.35 (d, 1H,
J = 3.20 Hz, ArH), 7.34–7.30 (m, 3H, ArH), 7.16 (d, 2H, J = 8.24 Hz,
ArH), 7.07 (d, 2H, J = 6.40 Hz, ArH), 5.33 (s, 2H, N-CH2), 3.98 (s,
4H, mor-CH2), 3.73 (s, 4H, mor-CH2), 2.55 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): d = 170.9, 165.1, 165.0, 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), 66.9 (O-CH2), 46.9 (N-CH2), 43.7 (N-CH2), 14.0
(CH3); EIMS, m/z: 478.3 (M++1); Anal. Calcd for C28H27N7O: C,
70.42; H, 5.70; N, 20.53, Found: C, 70.26; H, 5.62; N, 20.29.
By use of procedure of reaction 7, compound 18 using
N-methylpiperazine gave as white powder; (0.176 g, 77%); mp:
>300 °C; 1H NMR (400 MHz, CDCl3): d = 8.02 (d, 1H, J = 1.84 Hz,
ArH), 7.32–7.26 (m, 4H, ArH), 7.12 (d, 1H, J = 8.24 Hz, ArH), 7.05
(d, 2H, J = 7.80 Hz, ArH), 6.85 (br s, 1H, NH), 5.29 (s, 2H, N-CH2),
3.82–3.78 (m, 8H, mor-CH2, piperazine-CH2), 3.73 (t, 4H,
J = 8.72 Hz, mor-CH2), 2.55 (s, 3H, CH3), 2.44 (t, 4H, J = 10.08 Hz,
piperazine-CH2), 2.39 (s, 3H, N-CH3); 13C NMR (100 MHz, CDCl3):
d = 165.4, 165.1, 165.0, 164.5, 152.5, 152.1, 143.0, 142.7, 135.9,
134.4, 131.1, 129.0, 127.9, 126.2, 116.1, 110.6, 109.1 (ArC), 66.9
(O-CH2), 56.3 (N-CH2), 55.0 (N-CH2), 47.1 (N-CH2), 46.3 (N-CH2),
43.7 (N-CH2), 43.1 (N-CH3), 14.1 (CH3). EIMS, m/z: 500.3 (M++1).
Anal. Calcd for C27H33N9O: C, 64.91; H, 6.66; N, 25.23, Found: C,
64.70; H, 6.53; N, 25.31.
9.1.18. N-(2-Amino-ethyl)-N0-(3-benzyl-2-methyl-3H-benzimida-
zol-5-yl)-6-morpholin-4-yl-[1,3,5]triazine-2,4-diamine (19)
By use of procedure of reaction 7, compound 19 using
1,2-ethylenediamine gave as white powder; (0.172 g, 82%); mp:
>300 °C; 1H NMR (400 MHz, CDCl3): d = 8.05 (br s, 1H, NH),
7.31–7.26 (m, 5H, ArH), 7.11–7.08 (m, 3H, ArH), 5.29 (s, 2H,
N-CH2), 3.79 (s, 4H, mor-CH2), 3.72 (s, 4H, mor-CH2), 3.48 (t, 2H,
J = 5.48 Hz, aliphatic-CH2), 2.91 (t, 2H, J = 5.52 Hz, aliphatic-CH2),
2.55 (s, 3H, CH3), 1.65 (br s, 2H, NH2); 13C NMR (100 MHz,
CDCl3): d = 166.2, 165.8, 164.5, 152.4, 143.0, 135.9, 134.9, 132.9,
131.7, 129.1, 128.0, 126.2, 109.1 (ArC), 69.1 (O-CH2), 47.2
(N-CH2), 43.7 (N-CH2), 43.6 (N-CH2), 41.8 (N-CH2), 14.1 (CH3);
EIMS, m/z: 460.3 (M++1); Anal. Calcd for C24H29ON9: C, 62.73; H,
6.36; N, 27.43, Found: C, 62.33; H, 6.40; N, 27.87.
9.1.22. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-[4-(4-fluoro-
phenyl)-6-morpholin-4-yl-[1,3,5]triazin-2-yl]-amine (23)
By use of procedure of reaction 22, compound 23 using 4-fluor-
ophenyl boronic acid gave as white powder; (0.162 g, 71%); mp:
>300 °C; 1H NMR (400 MHz, CDCl3): d = 8.34 (t, 2H, J = 7.32 Hz,
ArH), 7.98 (br s, 1H, NH), 7.66 (d, 2H, J = 8.68 Hz, ArH), 7.32–7.26
(m, 4H, ArH), 7.05 (d, 4H, J = 5.96 Hz, ArH), 5.30 (s, 2H, N-CH2),
3.95 (s, 4H, mor-CH2), 3.72 (s, 4H, mor-CH2), 2.52 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3): d = 170.0, 165.1, 164.4, 152.0, 138.9,
137.7, 135.8, 134.0, 132.1, 132.0, 129.7, 129.2, 128.6, 128.5,
128.0, 125.8, 119.2, 115.7, 101.5 (ArC), 66.8 (O-CH2), 46.9
(N-CH2), 43.7 (N-CH2), 14.0 (CH3); EIMS, m/z: 496.3 (M++1); Anal.
Calcd for C28H26FN7O: C, 67.86; H, 5.29; N, 19.79, Found: C,
67.67; H, 4.99; N, 19.80.
9.1.19. 2-[4-(3-Benzyl-2-methyl-3H-benzimidazol-5-ylamino)-
6-morpholin-4-yl-[1,3,5]triazin-2-ylamino]-ethanol (20)
By use of procedure of reaction 7, compound 20 using 1,2-etha-
nolamine gave as white powder; (0.173 g, 85%); mp: >300 °C; 1H
NMR (400 MHz, CDCl3): d = 8.14 (br s, 1H, NH), 7.30–7.27 (m, 4H,
ArH), 7.04–7.02 (m, 4H, ArH), 6.40 (br s, 1H, NH), 5.21 (s, 2H,
N-CH2), 3.76–3.70 (m, 8H, mor-CH2), 3.63–3.62 (m, 2H, aliphatic-
CH2), 2.86 (t, 2H, J = 10.08 Hz, aliphatic-CH2), 2.54 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3): d = 166.8, 165.0, 152.5, 142.8, 135.8,
133.9, 131.8, 129.1, 128.0, 126.2, 116.6, 111.4, 109.1 (ArC), 69.1
(O-CH2), 66.9 (O-CH2), 47.2 (N-CH2), 43.7 (N-CH2), 43.6 (N-CH2),
14.1 (CH3); EIMS, m/z: 461.3 (M++1); Anal. Calcd for C24H28N8O2:
C, 62.59; H, 6.13; N, 24.33, Found: C, 62.50; H, 6.31; N, 24.47.
9.1.23. (1-Benzyl-2-methyl-1H-benzimidazol-5-yl)-[4-(4-chloro-
phenyl)-6-morpholin-4-yl-[1,3,5]triazin-2-yl]-amine (24)
By use of procedure of reaction 22, compound 24 using 4-chlor-
ophenyl boronic acid gave as white powder; (0.184 g, 78%); mp:
>300 °C; 1H NMR (400 MHz, CDCl3): d = 8.33 (t, 2H, J = 7.32 Hz,
ArH), 8.00 (br s, 1H, NH), 7.66 (d, 2H, J = 8.68 Hz, ArH), 7.34–7.26
(m, 3H, ArH), 7.15 (dd, 1H, 2J = 8.72 Hz, 3J = 1.84 Hz, ArH), 7.05 (d,
4H, J = 5.96 Hz, ArH), 5.30 (s, 2H, N-CH2), 3.95 (s, 4H, mor-CH2),
3.72 (s, 4H, mor-CH2), 2.52 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3): d = 170.0, 165.1, 164.4, 152.0, 138.9, 137.7, 135.8, 135.4,
134.0, 132.2, 132.1, 132.0, 129.7, 129.2, 128.6, 128.5, 128.0,
125.8, 119.2, 115.7, 101.5 (ArC), 66.8 (O-CH2), 46.9 (N-CH2), 43.7
(N-CH2), 14.1 (CH3); EIMS, m/z: 512.3 (M++1); Anal. Calcd for
9.1.20. N-(3-Benzyl-2-methyl-1H-benzimidazol-5-yl)-6-mor-
pholin-4-yl-N0-(2-morpholin-4-yl-ethyl)-[1,3,5]triazine-2,4-
diamine (21)
By use of procedure of reaction 7, compound 21 using
2-aminoethylmorpholine gave as white powder; (0.191 g, 79%);
mp: >300 °C; 1H NMR (400 MHz, CDCl3): d = 8.08 (s, 1H, NH),
7.30 (d, 4H, J = 7.36 Hz, ArH), 7.10–7.05 (m, 4H, ArH), 5.42 (br s,
C28H26ClN7O: C, 65.68; H, 5.12; N, 19.15, Found: C, 65.88; H,
5.21; N, 19.10.
Please cite this article in press as: Singla, P.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.012

P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
9.2. In vitro anticancer screening
(TGI) was calculated from Ti = Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the end
of the drug treatment as compared to that at the beginning) indi-
cating a net loss of cells following treatment was calculated from
[(Ti ꢀ Tz)/Tz] ꢁ 100 = ꢀ50. Values were calculated for each of these
three parameters if the level of activity was reached; however, if
the effect was not reached or was exceeded, the value for that
parameter was expressed as greater or less than the maximum
or minimum concentration tested.
Anticancer screening of compounds was carried out at National
Cancer Institute, Bethesda, MD, US, as per the standard procedure.
Compounds were submitted to National Cancer Institute (NCI) for
evaluation of their in vitro antitumor activities. Thirty one com-
pounds were evaluated against 60 cell lines at a single dose of
10ꢀ5 M which includes nine tumor subpanels and their output
was reported as a mean graph of the percent growth of treated
cells, and presented as percentage growth inhibition (GI%).
Compounds which exhibit significant growth inhibition were
evaluated against the 60 cell panel at five concentration levels.
The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum
9.3. DNA binding studies
Preparation of stock and ct-DNA solution: Stock solution of
compounds (10 mM) was prepared in AR grade DMSO. The DNA
binding experiments were carried out by making dilution of the
stock solution with phosphate buffer. Stock solution of ct-DNA
(Sigma product code D4522) was prepared by dissolving powdered
lyophilized DNA in phosphate buffer (10 mM, pH 7.0). The DNA
concentration was estimated from its absorbance intensity at
260 nm with a known molar absorption coefficient value of
6600 dm3 molꢀ1 cmꢀ1. The purity of DNA was established from
ratio of absorbance intensity at 260 nm and at 280 nm.
UV–Visible and fluorescence titrations: The titration experi-
ments were performed by varying the concentration of ct-DNA
and keeping the drug concentration between 10 and 40 lM. All
the UV–Visible spectra were recorded after equilibration of solu-
tion for 5 min. Fluorescence titration were carried out on Cary
Eclipse Fluorescence Spectrophotometer at ambient temperature.
A slit width of 10 nm was used with suitable kex. The titration
experiment was accomplished by varying the concentration of
and 2
l
M
L-glutamine. For a typical screening experiment, cells
were inoculated into 96 well microtiter plates in 100
lL at plating
densities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37 °C, 5% CO2, 95% air and
100% relative humidity for 24 h prior to addition of experimental
drugs. After 24 h, two plates of each cell line were fixed in situ with
TCA, to represent a measurement of the cell population for each
cell line at the time of drug addition (Tz). Experimental drugs were
solubilized in dimethyl sulfoxide at 400 fold the desired final max-
imum test concentration and stored frozen prior to use. At the time
of drug addition, an aliquot of frozen concentrate was thawed and
diluted to twice the desired final maximum test concentration
with complete medium containing 50 lg/mL gentamicin.
Additional four, 10-fold or 1/2log serial dilutions were made to
provide a total of five drug concentrations plus control. Aliquots
of 100
l
L of these different drug dilutions were added to the
L of medium,
DNA in cuvette (0.5–150 lM).
appropriate microtiter wells already containing 100
l
DNA thermal denaturation: DNA melting experiment was car-
ried out by monitoring the absorbance of ct-DNA at 260 nm at var-
ious temperature in the presence and absence of drug at 400 nm
with a ramp rate of 0.5 °C/min in a phosphate buffer (pH 7.0) on
a Shimadzu Spectrophotometer equipped with a Peltier thermo
regulator.
resulting in the required final drug concentrations. Following drug
addition, the plates were incubated for an additional 48 h at 37 °C,
5% CO2, 95% air, and 100% relative humidity. For adherent cells, the
assay was terminated by the addition of cold TCA. Cells were fixed
in situ by the gentle addition of 50 lL of cold 50% (w/v) TCA (final
concentration, 10% TCA) and incubated for 60 min at 4 °C. The
supernatant was discarded, and the plates were washed five times
with tap water and air dried. Sulforhodamine B (SRB) solution
Equation used for calculating binding constants: The
BenesiꢀHildebrand Equation equation is represented as follow:
1=ðAf ꢀ AobsÞ ¼ 1=ðAf ꢀ AfcÞ þ f1=½KðAf ꢀ AfcÞꢃg½ligandꢃ
(100 lL) at 0.4% (w/v) in 1% acetic acid was added to each well,
where K is the binding constant, Af is the absorbance of the free
host, Aobs is the absorbance observed, and Afc is the absorbance at
saturation.
and plates were incubated 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. Bound stain was sub-
sequently solubilized with 10 mM Trizma base, and the absorbance
was read on an automated plate reader at a wavelength of 515 nm.
For suspension cells, the methodology was the same except that
the assay was terminated by fixing settled cells at the bottom of
9.4. Dihydrofolate reductase inhibition assay
The dihydrofolate reductase inhibition assay was performed as
per the manual of the DHFR assay kit (Sigma product code CS0340).
All the dilutions were made in assay buffer, pH 7.5. 10 mM stock
solutions of dihydrofolic acid and NADPH in assay buffer were pre-
pared. Stock solutions of the test compounds with different con-
the wells by gently adding 50 lL of 80% TCA (final concentration,
16% TCA). Using the seven absorbance measurements [time zero,
(Tz), control growth, (C), and test growth in the presence of drug
at the five concentration levels (Ti)], the percentage growth was
calculated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as:
centrations were prepared in DMSO, and an amount of 20 lL of
each was taken to attain final concentration of 10ꢀ8, 10ꢀ7, 10ꢀ6
and 10ꢀ5 M in the respective wells of 96-well plate containing
assay buffer. An amount of 0.1 unit of DHFR as supplied in the
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢃ ꢁ 100
for concentrations for which Ti P Tz
½ðTi ꢀ TzÞ=Tzꢃ ꢁ 100
kit was diluted, and an amount of 20
used in each reaction. Each well of the 96-well plate was charged
l
L of its 3 ꢁ 10ꢀ3 unit was
with 157.8
lL of assay buffer. Then 1.2
lL of NADPH solution
for concentrations for which Ti < Tz.
was added to each well except 1A and 1B, and 20
lL of test com-
Three dose response parameters were calculated for each
experimental agent. Growth inhibition of 50% (GI50) was calculated
from [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢁ 100 = 50, which was the drug concen-
tration resulting in a 50% reduction in the net protein increase
(as measured by SRB staining) in control cells during the drug incu-
bation. The drug concentration resulting in total growth inhibition
pound (including methotrexate as positive control) was added to
each well except 1AꢀH. The reaction was started by the addition
of 1 lL of dihydrofolic acid to each well except 1C and 1D. 1G
and 1H contained 20
activity due to DMSO. The change in absorbance at 340 nm was
monitored as function of time. Percentage inhibition of
lL of DMSO to check any inhibition of enzyme
a
Please cite this article in press as: Singla, P.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.012

10
P. Singla et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
enzymatic activity was calculated after nullifying the effects of
NADPH, folate, and solvent. IC50 was calculated by plotting a graph
between percentage inhibition and the corresponding concentra-
tion of the compound using Graphpad Prism version 6.01.
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Compounds were built using the builder tool kit of the software
package Argus Lab 4.0.1.23 and energy minimized with semi-em-
pirical quantum mechanical method PM3. Crystal coordinates of
dihydrofolate reductase (DHFR) (PDB ID 3GHW) was downloaded
from protein data bank and in the molecule tree view of the soft-
ware, the monomeric structures of the crystal co-ordinate was
selected and the active site was defined as 15 Å around the ligand.
Validation of the docking programme was checked by docking the
known inhibitors of the respective enzymes in their binding sites.
The molecule to be docked in the active site of the enzyme was
pasted in the work space carrying the structure of the enzyme.
The docking programme implements an efficient grid based dock-
ing algorithm which approximates an exhaustive search within the
free volume of the S19 binding site cavity. The conformational
space was explored by the geometry optimization of the flexible
ligand (rings were treated as rigid) in combination with the incre-
mental construction of the ligand torsions. Thus, docking occurs
between the flexible ligand parts of the compound and enzyme.
The docking was repeated several times (approx. 10,000 iterations)
until no change in the position of the ligand and a constant value of
the binding energy was observed. The ligand orientation was
determined by a shape scoring function based on Ascore and the
final positions were ranked by lowest interaction energy values.
H-bond, hydrophobic and van der Waal’s interactions between
the respective compound and enzyme were explored. During com-
putational docking, a pose is typically generated, scored and com-
pared to the previous pose(s). The current pose is then accepted or
rejected on the basis of the score for that pose.
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Acknowledgments
K.P. thanks the SERC Fast Track Research Grant (SR/FT/CS-40/
2010), Department of Science and Technology, New Delhi. PS is
grateful for a DST/Inspire Fellowship (Fellow Code-IF110542).
Thanks are due to NCI, Bethesda, MD, US for anticancer screening
of compounds.
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27. Compounds were constructed and docked with builder tool kit of software
package ArgusLab 4.0.1 (www.arguslab.com).
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.03.012.
Please cite this article in press as: Singla, P.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.03.012