Exploring the potential of newly synthesized 4-methyl-6-morpholino-pyrimidine derivatives as antiproliferative agents

Article abstract of DOI:10.1039/c7nj04157h

In view of exploring the potential of pyrimidine derivatives as anticancer agents, a series of 4-methyl-6-morpholinopyrimidine derivatives was synthesised and characterised by NMR (¹H & ¹³C), SC-XRD and mass spectral analysis. The in vitro anticancer activity of these compounds was investigated using different human cancer cell lines, namely HeLa (cervix), NCI-H460 (lung), MCF-7 (breast), HepG2 (liver) and IMR-32 (brain). Compounds 4c and 5h exhibited potent anticancer activity in a dose-dependent manner as compared to other derivatives, with IC₅₀ values of 5.88 ± 1.22 and 6.11 ± 2.12 μM on HeLa and NCI-H460, cells respectively. The inhibitory effect of 4c and 5h on cancer cell proliferation was shown to be a consequence of reactive oxygen species (ROS) generation and subsequent induction of cellular apoptosis, as evidenced by an increase in hypodiploid (subG1) population, early apoptotic cell population, caspase-3/7 activity, loss of mitochondrial membrane potential and degradation of nuclear DNA. Furthermore, molecular docking studies revealed that 4c and 5h compounds bind to the ATP binding pocket of the mammalian target of rapamycin (mTOR). Based on our results, we conclude that 4-methyl-6-morpholinopyrimidine derivatives prevent cancer cell proliferation by inducing apoptosis and thus have potential to be further explored for anticancer properties.

Full text of DOI:10.1039/c7nj04157h

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Exploring the potential of newly synthesized 4ꢀmethylꢀ6ꢀmorpholinoꢀ
pyrimidine derivatives as antiproliferative agents
Supreet Gaonkara,1, Mohammed Azharuddin Savanurb,c,1 , Manjunath G. Sunagara, Bijesh
Puthusserid , Narahari Deshapandea, Afra A. Nadafa, Imtiyaz Ahmed M. Khazia,*
a Department of Studies in Chemistry, Karnatak University, Dharwad 580003, Karnataka,
India.
b Department of Biochemistry, Karnatak University, Dharwad 580003, Karnataka, India.
c Present Address: Indian Institute of Science, Bangalore 560012, India.
d CSIRꢀCentral, Food Technological Research Institute, Mysore 570020, India.
1 These authors contributed equally to this work
*Corresponding author , Imtiyaz Ahmed M. Khazi; Email: drimkorgchem@gmail.com
Abstract
In view of exploring the potential of pyrimidine derivatives as anticancer agents, a
series of 4ꢀmethylꢀ6ꢀmorpholinopyrimidine derivatives were synthesised and characterised by
NMR (1H & 13C), SCꢀXRD and Mass spectral analysis. The in vitro anticancer activity of
these compounds was investigated using different human cancer cell lines namely HeLa
(cervix), NCIꢀH460 (lung), MCFꢀ7(breast), HepG2 (liver) and IMRꢀ32 (brain). Compounds
4c and 5h exhibited potent anticancer activity in dose dependent manner as compared to other
derivatives, with an IC50 values of 5.88 ± 1.22 and 6.11± 2.12 µM on HeLa and NCIꢀH460
cells respectively. The inhibitory effect of 4c and 5h on cancer cell proliferation was shown
to be a consequence of reactive oxygen species (ROS) generation and subsequent induction
of cellular apoptosis, as evidenced by increase in hypodiploid (subG1) population, early
apoptotic cell population, caspaseꢀ3/7 activity, loss of mitochondrial membrane potential and
degradation of nuclear DNA. Furthermore, molecular docking studies revealed that 4c and 5h
compounds binds to ATP binding pocket of mammalian target of rapamycin (mTOR). Based
on our results we objectify that 4ꢀmethylꢀ6ꢀmorpholinopyrimidine derivatives prevent cancer
cell proliferation by inducing apoptosis and thus have potential to be further explored for
anticancer properties.
Keywords: Morpholinopyrimidine; Antiꢀproliferative; Cytotoxic; Apoptosis; mTOR

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1. Introduction
Cancer is a multifactorial disease with high rate of mortality. Lung, liver, prostate,
stomach and colorectal cancer are the most common types of cancer in men, while breast,
cervix, colorectal, lung and stomach cancer are common among women.1 Development of
cancer in humans is a multistep process that includes generating persistent growth signals
and insensitivity to antiꢀgrowth signals, evasion of apoptosis, unlimited replication
potential, angiogenesis, invasion and metastasis.2 The discovery of targetedꢀdrug
therapies that intervene the cancer development process has patently impeded
malignancies.3 However, the alarming rise of new types of cancer and advanced
metastasized cancer remains untreatable. Hence, a continued search for development of
selective, more potent and less toxic chemotherapeutic agents is needed for the treatment of
cancer.
Organic moieties specifically those containing a hetero atom in their aromatic nucleus
have added to the growth in the library of bioactive compounds and have shown wide
applications in medicinal chemistry fields. According to the FDA (Food and Drug
Administration) database, nitrogen containing heterocyclic compounds are seen in
approximately 60% of the small molecule drugs which proves the significance of nitrogen
based heterocycles.4 Pyrimidines are the most important six membered aromatic heterocyclic
compounds widely distributed in nature as nucleotides, thiamine and alloxan, whereas
barbiturates are one of their earliest synthetic forms. There are numerous reports available in
literature, which has shown that pyrimidine libraries against some biological targets have
contributed significantly to the development of pharmacologically active agents.5ꢀ7 Synthetic
pyrimidine derivatives exhibits remarkable pharmacological activities and form a component
in a number of useful drugs, which are associated with many biological and therapeutic
activities.8 In particularly, pyrimidine derivatives are found to act as anticancer,9 antiꢀHIV,10
antibacterial,11 antihypertensive,12 anticonvulsant,13 antitubercular,14 analgesic15 and
antiplasmodial agents.16 Among the literature reports of pyrimidines, anticancer activity is
more extensively studied. Because of the vast diversity in its chemistry, researchers are
working in the synthesis of substituted pyrimidines as anticancer agents. Some of the
pyrimidine based anticancer drugs approved by FDA
Osimeritinib, Idelalisib, Afatinib, Palbocilib, Ceritinib etc.,
are 5ꢀFlurouracil, Tegafur,
Morpholine is the most commonly used heterocyclic secondary amine substituent in
drugs and pharmaceutically active compounds.17 Morpholine and pyrimidine hybrids gained

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attention of medicinal chemist because of their therapeutic application as antitumor,18
antimicrobial,19 antiꢀinflammatory,20 antisenility21 agents.22
and antimalarial
Morpholinopyrimidines are biologically important molecules, mainly studied as
PI3K/Akt/mTOR pathway inhibitors.23ꢀ25 Many of these PI3K/mTOR inhibitors share
morpholinopyrimidine as common structural unit such as Apitolisib (GDCꢀ0980),26 Pictilisib
(GDCꢀ0941),27 PIꢀ103,28 GNEꢀ47729 and so on (Fig. 1).
In the present study we report synthesis and cytotoxicity of 4ꢀmethylꢀ6ꢀ
morpholinopyrimidine derivatives against different cancer cell lines. In addition, this report
also explains the mechanism of morpholinopyrimidine derivatives induced cell death by
generation of ROS and subsequent induction of cellular apoptosis, as well as their interaction
with mTOR as possible target.
2. Result and Discussion
2.1 Chemistry
The synthetic routes for 4ꢀmethylꢀ6ꢀmorpholinopyrimidine derivatives (4aꢀ4h & 5aꢀ
5h) are outlined in Scheme 1. 2,4ꢀdichloroꢀ6ꢀmethylpyrimidine (2) was synthesised by
treating 6ꢀmethylpyrimidineꢀ2,4(1H,3H)ꢀdione (1) and phosphorusoxychloride under reflux
temperature followed by the neutralisation with sodium bicarbonate. The key intermediate 2ꢀ
chloroꢀ4ꢀmethylꢀ6ꢀmorpholinopyrimidine (3) was synthesised by reacting 2 with morpholine
and potassium carbonate in dioxane at room temperature. Bromination of 3 was done by Nꢀ
bromosuccinimide in benzene to afford 5ꢀbromoꢀ2ꢀchloroꢀ4ꢀmethylꢀ6ꢀmorpholinopyrimidine
(4). The target compounds 4(aꢀh) and 5(aꢀh) are synthesised with good yield by
nucleophilic substitution reaction of various amines (aꢀh) with either 4 or 3 respectively.
Stuctures of the synthesized compounds were characterised by 1H NMR, 13C NMR and Mass
spectral analysis. Structure of compound 4a was supported by NMR spectral studies, the
characteristic singlet at 8.89 ppm and a broad singlet at 5.71 ppm for two protons due to C8ꢀ
H of purine and C2ꢀNH2 of purine respectively. Further the formation of compound 4a was
confirmed by LCꢀMS data. The 1H NMR spectrum of compound 4c shows three
characteristic singlets at 8.53 ppm 7.77 ppm and 7.10 ppm due to C2ꢀH, C5ꢀH and C4ꢀH of
imidazole respectively. Two triplets at 3.83 ppm for four protons and 3.67 ppm for four
protons corresponding to two CH2 groups next to oxygen atom and two CH2 group next to
nitrogen atom of morpholine respectively, it is further supported by 13C NMR and Mass
spectral data. Structure of the compound 4h was confirmed by single crystal Xꢀray analysis.

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A characteristic singlet at 6.20 ppm due to C5ꢀH of pyrimidine supports the formation of
compound 5c and it is confirmed by 13C NMR and Mass spectral data.
O
O
N
O
O
Cl
N
N
N
H
POCl3
Reflux
NBS, Benzene
2 hr.
Br
NH
N
N
N
Dioxane
K2CO3
N
H
O
N
Cl
Cl
N
Cl
RT, Overnight
3
1
2
1
4
1
o
r
o
r
2
2
a
m
a
m
i
n
i
e
n
e
O
N
O
N
Br
N
N
N
R
N
R
5(a-h)
4(a-h)
NH2
NH
N
N
N
N
N
N
N
N
R=
S
NH
N
H2N
N
OH
CN
CH3
O
NH2
O
g
a
c
e
f
b
d
h
Scheme 1: Synthesis of 4ꢀmethylꢀ6ꢀmorpholinopyrimidine derivatives
2.2. Single crystal X-ray analysis
A suitable single crystal was collected through the polarising microscope and
mounted on the 'Bruker APEXꢀII CCD' diffractometer system is equipped with micro focus
sealed tube Mo source, CCD area detector. Crystal was kept at 298K (2) during the data
collection with scan width of 0.5mm and distance of 45mm from crystal to detector. The
structure (Fig. 2) was solved using the Olex2 with the XT,30 using Intrinsic Phasing and
refined with the ShelXL refinement package using Least Squares minimization.31 The
molecule crystallizes in the monoclinic crystal system having a centrosymmetric space group
i.e. P21/n and one molecule in the asymmetric unit. In the crystal structure Br…Br (3.590 Å)
Br…π (3.491 Å) and also CH… π (2.892 Å) interaction makes the molecule more stable (Fig.
3). Crystal data are listed in the Table 1.

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Table 1: Crystal data and structure refinement of compound 4h.
Empirical formula
C18H23BrN4O2
Formula weight
Temperature/K
Crystal system
Space group
a/Å
b/Å
c/Å
α/°
407.31
293(2)
Monoclinic
P21/n
18.8570(6)
4.98220(10)
21.8645(7)
90
β/°
γ/°
115.2770(10)
90
1857.48(9)
4
1.457
2.232
Volume/Å3
Z
ρcalcg/cm3
ꢁ/mmꢀ1
F(000)
840.0
0.26 × 0.25 × 0.24
MoKα (λ = 0.71073)
Crystal size/mm3
Radiation
2Θ range for data collection/°8.264 to 49.998
Index ranges
ꢀ22 ≤ h ≤ 22, ꢀ5 ≤ k ≤ 5, ꢀ25 ≤ l ≤ 25
Reflections collected
Independent reflections
Data/restraints/parameters
Goodnessꢀofꢀfit on F2
48066
3212 [Rint = 0.0341, Rsigma = 0.0137]
3212/0/228
1.131
Final R indexes [I>=2σ (I)] R1 = 0.0393, wR2 = 0.1106
Final R indexes [all data] R1 = 0.0437, wR2 = 0.1144
Largest diff. peak/hole / e Åꢀ3 0.48/ꢀ0.90
CCDC number
1478498
2.3. In vitro Anticancer activity
2.3.1. Morpholinopyrimidine compounds inhibit proliferation of cancer cells
Cytotoxicity of newly synthesized 4ꢀmethylꢀ6ꢀmorpholinopyrimidine derivatives was
determined in-vitro on HeLa, NCIꢀH460, MCFꢀ7, HepG2 and IMRꢀ32 cell lines by using
MTT assay. The IC50 (µM) values (concentration required to inhibit 50% of cancer cell
proliferation) of the tested compounds and the standard reference doxorubicin are listed in
Table 2. The results of cell viability assay showed strong growth inhibitory effect of
Morpholinopyrimidine compounds on cancer cells in dose dependent manner. Compounds 4c
and 5h showed higher activity as compared to other derivatives, with an IC50 values of 5.88 ±
1.22 and 6.11± 2.12 µM on HeLa and NCIꢀH460 cells respectively. The dose dependent
cytotoxic effect of 4c and 5h was observed in HeLa and NCIꢀH460 cells, where at 20µM

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compound 4c (Fig. 4A) caused 86.2 ± 3.44% (P<0.0001) and 79.3 ± 4.2% (P<0.0001), and
compound 5h (Fig. 4B) caused 75.2 ± 3.8 % (P<0.0001) and 84.3 ± 3.34% (P<0.0001)
growth inhibition in HeLa and NCIꢀH460 cells respectively, suggesting the cytotoxic effect
of Morpholinopyrimidine compounds (4c and 5h) is dose dependent. Considering the potent
cytotoxic effect of 4c and 5h on HeLa and NCIꢀH460 cells, respectively, the effect of
compound 4c and 5h on morphology of cancer cells was determined. Cancer cells in control
group had distinct, well defined morphology with clear skeletons; In contrast cells treated
with 4c and 5h compounds had indecisive morphology with indistinct skeletons and appeared
to be dead as evidenced by phase contrast images (Fig. 4C), demonstrating the cytotoxic
effect of these compounds.
From table 2 it is evident that compounds comprising imidazole (4c) and 2ꢀ(3ꢀ
methoxyphenyl) ethanamine (5h) at Cꢀ2 position of the pyrimidine are more potent as
compared to remaining derivatives. The antiproliferative activity of compound 4c and 5c in
HeLa cell line is attributed due to the presence of imidazole ring. Since imidazole ring has
structural resembles with histidine, it can easily binds with proteins compared to other
heterocyclic rings. Many of the anticancer compounds are having imidazole skeleton as
structural backbone.32,33 Compound 5h and 4h have shown good and moderate activity
against NCIHꢀ460 cell lines respectively. The compounds having ethanamine and
ethylenediamine linkage with conformational flexibility have been important pharmacophore
in many biologically significant molecules. This could be the possible reason for the
enhanced activity.34 Compounds 4a, 4b, 5a & 5b showed low solubility and are less potent
than the remaining derivatives of morpholinopyrimidine (4d, 4e, 4f, 4g, 4h, 5c, 5d, 5e, 5f, 5g)
which are moderately active.
Table 2 : Antiꢀproliferative activity of Morpholino pyrimidine compounds on HeLa, NCIꢀ
H460, MCFꢀ7, HepG2 and IMRꢀ32 cell lines was determined by MTT assay and the IC50
values were calculated based on results obtained at 24ꢀh time point.
IC50 values (µM) of Morpholino pyrimidine derivatives
Compounds
HeLa
NCIꢀH460
MCFꢀ7
HepG2
IMRꢀ32
4a
4b
4c
>20
>20
>20
>20
>20
>20
>20
>20
16.92 ± 3.30
19.88 ± 3.41
13.63 ± 2.94
11.34 ± 2.63 12.99 ± 2.30 14.12 ± 3.78
5.88 ± 1.22

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4d
4e
4f
13.69 ± 1.93
>20
16.46 ± 1.02 15.84 ± 2.36 16.05 ± 1.40
>20
12.61 ± 3.78 16.84 ± 3.63 15.69 ± 0.89 14.35 ± 1.91
16.72 ± 3.68
12.72 ± 2.12 15.27 ± 2.69 14.93 ± 4.32
>20 >20 >20
>20
>20
4g
4h
5a
5b
5c
5d
5e
5f
>20
17.28 ± 4.12
16.04 ± 0.93 12.98 ± 2.68 17.34 ± 2.38 16.44 ± 3.07
18.03 ± 2.36
>20
>20
>20
>20
19.81 ± 2.08
>20
>20
>20
>20
15.74 ± 0.93
12.09 ± 1.68 14.77 ± 2.89 13.52 ± 2.10 12.84 ± 3.27
17.46 ± 3.02 16.37 ± 2.16 15.92 ± 1.68 14.56 ± 1.74
16.69 ± 2.92 10.28 ± 3.01 17.17 ± 4.13 13.91 ± 2.97
15.53 ± 1.73
>20
14.49 ± 1.89
>20
17.63 ± 4.37 16.19 ± 1.05 16.60± 1.36
15.96 ± 2.03 14.06 ± 2.32 16.11 ± 2.60 18.41 ± 1.09
10.02 ± 1.39 11.69 ± 3.62 13.32 ± 2.34
>20
5g
5h
14.56 ± 1.92
6.11 ± 2.12
2.3.2. Compounds 4c and 5h induce accumulation of HeLa cells in hypodiploid (subG1)
phase of cell cycle.
The effects of cytotoxic compounds are mainly exerted by arresting the cells at
specific phase of a cell cycle, thus preventing cellular proliferation and inducing cell death.35ꢀ
37 The population of cells undergoing apoptosis are represented as the hypodiploid population
in the subG1 phase.38,39 Distribution of cells in the different phases of cell cycle was
determined by exposing HeLa and NCIꢀH460 cells to 4c and 5h compounds respectively at
different concentrations, followed by propidium iodide (PI) staining and flow cytometry
analysis. Cells treated with compounds 4c and 5h showed a dose dependent increase in hypoꢀ
diploid population, where treatment with 10 µM of compounds resulted in 27.3% and 28.6%
respectively, which increased to 39.7% and 40.9%, respectively at 20 µM as compared to
1.5% and 1.8% respectively in control groups, suggesting that these molecules promote cell
death through induction of apoptosis (Fig. 5A). The percentage of cell populations in each
phase of cell cycle is represented in the bar diagram (Fig. 5B and 5C).
2.3.3. Induction of apoptosis by compound 4c and 5h.
Phosphatidylserine translocation to the outer surface of plasma membrane is a
hallmark of apoptosis,40,41 which can be detected using AnnexinꢀV.42,43 The potential of
compound 4c and 5h to induce apoptosis was determined by AnnexinꢀV and PI staining,
distinguishes viable, early and late apoptosis or dead cells. HeLa and NCIꢀH460 cells

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exposed to 4c and 5h compounds at different concentration showed dose dependent increase
in early and late apoptotic population. As shown in Fig. 6A, a significant increase in early
apoptotic cells (AnnexinꢀV positive) 52.7% and 50.2% was observed after treatment with 4c
and 5h compounds respectively, as compared to 0.5% and 1.1% in control groups,
respectively. There was also a dose dependent increase in late apoptotic cells (AnnexinꢀV and
PI positive) 32.2% and 33.8% respectively, indicating that these compounds induces cellular
apoptosis in a dose dependent manner. Percentage of cells in four different quadrants as
shown in Fig. 6B and 6C was represented as bar graph.
2.3.4. Intracellular ROS production by compound 4c and 5h.
Excessive amount of ROS can cause oxidative damage to biomolecules, inducing
apoptosis and cell death.44,45 Thus, compounds that exhibit a potent ROS generating ability in
cancer cells show promising anticancer activity.46ꢀ48 The ability of compound 4c and 5h to
generate ROS in cancer cells was determined by staining with DCFDAꢀH2, a nonꢀfluorescent
dye which upon oxidation by ROS emits green fluorescence. Results of fluorescence
microscopy revealed that cells treated with 4c and 5h showed increase in green fluorescence
as compared to control cells, indicating that these compounds promote ROS generation and
induce apoptosis in cancer cells (Fig. 8A).
2.3.5. Compound 4c and 5h induce loss of mitochondrial membrane potential.
Increase in intracellular ROS causes mitochondrial damage and loss in mitochondrial
membrane potential, further leading to apoptosis.49,50 Since, compound 4c and 5h induced
intracellular ROS production, the detrimental effects of elevated ROS levels on mitochondria
were analysed by measuring the changes in mitochondrial membrane potential. The effect of
4c and 5h on the mitochondrial membrane potential was determined by staining cells with Jcꢀ
1 dye, which accumulates in intact mitochondria emitting red fluorescence. The accumulation
of JCꢀ1 is membrane potential dependent and can be explored to measure changes in
mitochondrial polarisation. During apoptosis, mitochondrial depolarisation leads to collapse
of mitochondrial membrane potential, where JCꢀ1 accumulates in cytoplasm emitting green
fluorescence.51 An increase in the number of cells which are positive for green fluorescence
after treatment with 4c and 5h compounds at various concentrations (0ꢀ20 µM) was observed
(Fig. 7A), suggesting that the Compound 4c and 5h induced cell death involves
mitochondrial depolarisation associated apoptosis. The percentage of cell populations with

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high and low mitochondrial membrane potential are represented in the bar diagram (Fig. 7B
and 7C).
2.3.6. Activation of effector caspase-3/7 by Compound 4c and 5h.
The ultimate consequence of elevated ROS is oxidative stress mediated induction of
apoptosis.52,53 As the activation of effector caspaseꢀ3/7 brings about the characteristic
changes associated with apoptosis like DNA condensation and fragmentation, the potential of
compound 4c and 5h to activate effector caspases was determined using appropriate
substrates provided in caspaseꢀactivity detection kit. As shown in Fig. 8B, a significant
increase in caspase 3/7 activity (1.96 and 3.25 fold) was observed in cells treated with 4c and
5h compounds respectively when compared to untreated control, indicating that cell death
caused by these compounds is linked to induction of cell apoptosis.
2.4. Compound 4c and 5h causes chromatin condensation and DNA fragmentation
Nuclear shrinkage, chromatin condensation and fragmented DNA, are the remarkable
features of cellular apoptosis. Antiꢀcancer drugs are known to induce DNA fragmentation by
activation of specific endonucleases which leads to cancer cell death.54,55 As compound 4c
and 5h induced apoptotic cell death in cancer cells, we further investigated the ability of these
compounds to condense chromatin and fragment DNA, in order to determine their
mechanism to induce cell death. As shown in Fig. 8D, DAPIꢀstaining confirmed significant
chromatin condensation in Morpholino pyrimidine compounds treated cells, suggesting
induction of apoptosis. DNA strand breaks in cells exposed to 4c and 5h compounds was
analysed by agarose gel electrophoresis, where the treatment of 4c and 5h resulted in a
fragmented DNA ladder formation in a doseꢀdependent manner (Fig. 8C), further supporting
the evidence that these compounds induced cell death via induction of apoptosis.
2.5 Compound 4c and 5h binds to the ATP pocket of the mTOR
The ligands, 4c and 5h, docked into the catalytic domain of the mTOR, and the best
docked conformation is provided in (Fig. 9). Molecule 4c was found to have ligandꢀ
interactions with the amino acids Asn247, Ala311, Ile298 and Glu296 of the modelled
mTOR. The hydrophobic morpholino moiety of the 4c ligand was found to fit in the
hydrophobic pocket of the mTOR binding sites constituted by Ile298, Pro309, Leu306,
Phe301, Ala 311, Leu312 and Leu 250. Whereas, the hydrophilic pyrimidine end of the 4c
molecule was aligned towards the hydrophilic pocket constituted by the amino acids Lys256,

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Asn254, Met251, Glu310, Arg249 and Trp248 of the pharmacophore. Molecule 5h has
shown interactions with Glu9, Asp10, Gln13, Asp14, Asp176 and Asp252. The ligand, 5h,
was found to have its hydrophobic morpholine end docked in the hydrophobic pocket of the
binding site mainly constituted by the Val183, Gly178, Phe240, Leu246 and Gly291 of the
pharmacophore model. Whereas, the hydrophilic pyrimidine moieties of the 5h ligand was
aligned towards the hydrophilic pocket constituted by the amino acids Glu13, Glu9, His8,
Asp10, Arg12, Thr292, Trp248 and Asp176 of the mTOR catalytic domain. Previous studies
for screening mTOR inhibitors have noted the binding of ligands in the same binding site
with similar attributes.56ꢀ58 They have pointed out the importance of hydrogen bond acceptor
ability of bridged morpholine for the high activity of these inhibitors. Our study shows the
structural features for the activity of morpholinopyramidine as mTOR kinase inhibitors. The
study also shows the possible sites of modifications for improving the activity of these
inhibitors.
3. Conclusion
In summary, a series of sixteen 4ꢀmethylꢀ6ꢀmorpholinopyrimidine derivatives were
synthesized and their cytotoxic potential was evaluated using different human cancer cells.
Within the series, compound 4c and 5h exhibited strong growth inhibitory effect in a dose
dependent manner with IC50 values of 5.88 ± 1.22 and 6.11± 2.12 µM on HeLa and NCIꢀ
H460 cells respectively. The structure and activity relationship analysis revealed that
compounds consisting imidazole and 2ꢀ(3ꢀmethoxyphenyl) ethanamine at Cꢀ2 position of the
pyrimidine are more potent and showed pronounced cytotoxicity. Therefore, compound 4c
and 5h were selected for further studies to determine the mechanism of action. The cell cycle
analysis, annexinꢀV/PI assay, assessment of caspaseꢀ3/7 activity, evaluation of mitochondrial
membrane potential and DNA damage studies were carried out to determine the mechanistic
details of morpholinopyrimidine derivatives induced cancer cell death. The results of these
studies indicate that the observed antiꢀproliferative activity of 4c and 5h compounds on
cancer cells is a consequence of cellular apoptosis induction. Furthermore, molecular docking
of 4c and 5h compounds with mTOR shows that 4c and 5h compounds bind to ATP binding
pocket of the kinase. Based on the results obtained we conclude that 4ꢀmethylꢀ6ꢀ
morpholinopyrimidine derivatives can be further explored for anticancer properties and will
be studied as plausible mTOR inhibitors.

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4. Experimental
4.1 Chemistry
4.1.1. Materials and Method
All the solvents and reagents are of analytical grade and were used directly without
further purification. Thin layer chromatography (TLC) technique was employed to monitor
the progress of reaction. Merck Silica gel 60 F254 TLC plates were used and spots were
visualized using ultraviolet light (254 nm) or iodine chamber. Column chromatography was
carried out with silica gel 60 (100ꢀ200 mesh). Melting points were determined by open
1
capillary method and are uncorrected. H NMR and 13C NMR spectra were recorded on
Bruker 400 MHz and 101 MHz FT NMR spectrometer respectively, in DMSOꢀD6 and CDCl3
by using tetramethylsilane (TMS) as internal standard. Chemical shifts are expressed in ppm
(δ) and multiplicities of NMR signals are assigned as s (singlet), d (doublet), t (triplet), q
(quartet), bs (broad singlet), dt (doublet of triplet), tt (triplet of triplet), dq (doublet of
quartet), m (multiplet, for unresolved lines), etc. Mass spectra were recorded using Shimadzu
GCMSꢀQP2010S
4.1.2. General procedure for synthesis of (4a & 4c-4h)
A mixture of 5ꢀbromoꢀ2ꢀchloroꢀ4ꢀmethylꢀ6ꢀmorpholinopyrimidine (4) (1g, 0.00342
mol), anhydrous potassium carbonate (1.42g, 0.0102 mol) and primary or secondary amines
(0.00342 mol) in dry DMF was refluxed for about 8–10 h. Completion of the reaction was
monitored by thin layer chromatography (TLC). The reaction mixture was cooled and poured
in crushed ice. The resulting precipitate was filtered, washed with water, dried over reduced
pressure and recrystallised from ethanol.
4.1.2.1. 9-(5-bromo-4-methyl-6-morpholinopyrimidin-2-yl)-9H-purine-2,6-diamine (4a)
White solid (90% yield); mp: 266ꢀ270°C; 1H NMR (400 MHz, dmsoꢀd6) δ 8.89 (s, 1H), 5.71
13
(s, 2H), 3.70 (bs 10H), 2.52 (s, 3H); C NMR (101 MHz, CDCl3) δ 167.68, 164.10, 159.34,
156.08, 153.24, 150.22, 125.95, 115.28, 101.65, 65.98, 47.84, 25.38; LCMS: m/z 406.025.
4.1.2.2. 5-bromo-2-(1H-imidazol-1-yl)-4-methyl-6-morpholinopyrimidine (4c)
White solid (92% yield); mp: 96ꢀ100°C; 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 7.77 (s,
1H), 7.10 (s, 1H), 3.83 (t, J= 8.1 Hz 4H), 3.67 (t, J= 7.8 Hz 4H), 2.57 (s, 3H); 13C NMR (101

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MHz, CDCl3 ) δ 168.57, 163.70, 151.57, 136.28, 130.42, 116.68, 103.94, 66.62, 48.94, 25.42;
GCMS: m/z 323.
4.1.2.3. 5-bromo-4-methyl-6-morpholino-N-(thiophen-2-ylmethyl)pyrimidin-2-amine (4d)
White solid (84% yield); mp: 106ꢀ110°C; 1H NMR (400 MHz, CDCl3 ) δ 7.34 (d, J = 7.9 Hz
1H), 6.28 (t, J = 8.2 Hz 1H), 6.14 (d, J = 7.8 Hz 1H), 4.79 (bs, 1H), 4.18 (d, J = 7.1 Hz, 2H),
3.71 – 3.67 (m, 4H), 3.43 – 3.38 (m, 4H), 2.46 (s, 3H); 13C NMR (101 MHz, CDCl3 ) δ
168.93, 162.06, 159.36, 148.56, 146.83, 119.85, 114.72, 98.46, 66.37, 50.73, 44.45, 25.39;
GCMS: m/z 368
4.1.2.4. 2-((5-bromo-4-methyl-6-morpholinopyrimidin-2-yl)(methyl)amino)ethanol (4e)
1
White solid (78% yield); mp: 60ꢀ64°C; H NMR (400 MHz, CDCl3 ) δ 3.85ꢀ3.82 (m, 2H),
3.80 – 3.76 (m, 4H), 3.73 (bs 1H) 3.70 – 3.67 (m, 2H), 3.49 – 3.45 (m, 4H), 3.14 (s, 3H), 2.38
(s, 3H); 13C NMR (101 MHz, CDCl3) δ 166.66, 163.19, 160.22, 95.40, 66.89, 63.44, 53.04,
48.70, 37.11, 25.43; GCMS: m/z 330
4.1.2.5. 1-(5-bromo-4-methyl-6-morpholinopyrimidin-2-yl)piperidine-4-carbonitrile (4f)
White solid (83% yield); mp: 87ꢀ91°C; 1H NMR (400 MHz, ) δ 3.86 (t , J = 7.6 Hz, 4H),
3.64 – 3.58 (m, 4H), 3.26 – 3.21 (m, 2H), 2.95ꢀ2.90 (m, 2H), 2.51ꢀ 2.46 (m, 1H), 2.26 (s, 3H),
1.74ꢀ1.70 (m, 2H), 1.43ꢀ1.37 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 167.83, 164.53,
162.56, 118.39, 96.73, 65.98, 45.37, 44.92, 43.08, 29.17, 25.33; GCMS: m/z 365
4.1.2.6. 1-(5-bromo-4-methyl-6-morpholinopyrimidin-2-yl)piperidine-4-carboxamide (4g)
1
White solid (88% yield); mp: 201ꢀ205°C; H NMR (400 MHz, dmsoꢀd6) δ 7.13 (s, 1H), 6.69
(s, 1H), 4.06 – 4.01 (m, 2H), 3.71 (t , J = 8.1 Hz, 4H), 3.53 – 3.48 (m, 4H), 3.02ꢀ2.98 (m,
2H), 2.78ꢀ 2.73 (m, 1H), 2.49 (s, 3H), 1.83ꢀ1.77 (m, 2H), 1.39ꢀ1.33 (m, 2H); 13C NMR (101
MHz, dmsoꢀd6) δ 178.15, 166.18, 162.84, 160.93, 99.82, 67.29, 44.15, 43.27, 41.60, 27.73,
24.57; LCMS: m/z 384
4.1.2.7. N-(3-methoxyphenethyl)-5-bromo-4-methyl-6-morpholinopyrimidin-2-amine (4h)
1
White solid (91% yield); mp: 99ꢀ103°C; H NMR (400 MHz, CDCl3 ) δ 7.24 – 7.18 (m, 1H),
6.78 (m, 2H), 6.74 (s, 1H), 4.97 (bs, 1H), 3.81 – 3.77 (m, 7H), 3.59 (dt, J = 13.5, 6.9 Hz, 2H),
3.52 – 3.46 (m, 4H), 2.84 (t, J = 7.1 Hz, 2H), 2.39 (s, 3H); 13C NMR (101 MHz, CDCl3 ) δ

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166.91, 163.96, 159.82, 159.76, 141.00, 129.65, 121.24, 114.72, 111.62, 96.75, 66.96, 55.23,
49.05, 42.97, 36.08, 25.40; GCMS: m/z 406
4.1.3. Synthesis of 1-(5-bromo-4-methyl-6-morpholinopyrimidin-2-yl)-1H-indole (4b)
To a 0 °C solution of indole (0.4g, 0.00342 mol) in 10 mL of DMF was added sodium
hydride (0.123g, 0.00513 mol, 60% dispersion in oil), stirred at 0°C for 30 min, then 5ꢀ
bromoꢀ2ꢀchloroꢀ4ꢀmethylꢀ6ꢀmorpholinopyrimidine (4) (1g, 0.00342 mol) was added, and the
reaction mixture heated to 140°C for 2 h. The reaction mixture was cooled and diluted with
water, the solid separated was filtered and recrystallised from alcohol.
1
White solid (75% yield); mp: 182ꢀ186°C; H NMR (400 MHz, dmsoꢀd6) δ 8.62 (d, J = 8.2
Hz, 1H), 8.17 (d, J = 3.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.17 (t, J
= 7.2 Hz, 1H), 6.71 (d, J = 2.9 Hz, 1H), 3.75 (s, 4H), 3.61 (s, 4H), 2.56 (s, 3H); 13C NMR
(101 MHz, dmsoꢀd6); 168.31, 163.24, 158.99, 143.43, 130.68, 125.05, 123.89, 121.72,
114.09, 112.33, 101.21 98.73, 66.86, 49.69, 25.33; GCMS: m/z 372
4.1.4. General procedure for synthesis of (5a & 5c-5h)
A mixture of 2ꢀchloroꢀ4ꢀmethylꢀ6ꢀmorpholinopyrimidine (3) (1g, 0.00469 mol), anhydrous
potassium carbonate (1.94g, 0.0141 mol) and primary or secondary amines (0.00469 mol) in
dry DMF was refluxed for about 8–10 h. Completion of the reaction was monitored by thin
layer chromatography (TLC). The reaction mixture was cooled and poured in crushed ice.
The resulting precipitate was filtered, washed with water, dried over reduced pressure and
recrystallised from ethanol.
4.1.4.1. 9-(4-methyl-6-morpholinopyrimidin-2-yl)-9H-purine-2,6-diamine (5a)
White solid (89% yield); mp: 248ꢀ252°C; 1H NMR (400 MHz, dmsoꢀd6) δ 8.94 (s, 1H), 5.91
(s, 1H), 5.68 (bs, 2H), 3.78ꢀ3.72 (m 4H), 3.60ꢀ3.55 (m, 6H), 2.49 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 167.32, 163.95, 158.78, 156.47, 152.89, 149.71, 124.08, 115.20, 100.16,
66.05, 48.48, 25.49; GCMS: m/z 327
4.1.4.2. 2-(1H-imidazol-1-yl)-4-methyl-6-morpholinopyrimidine (5c)
1
White solid (91% yield); mp: 70ꢀ74°C; H NMR (400 MHz, CDCl3 ) δ 8.50 (s, 1H), 7.80 (s,
1H), 7.07 (s, 1H), 6.20 (s, 1H), 3.80 – 3.76 (m, 4H), 3.64 (t, J = 6.6 Hz, 4H), 2.36 (s, 3H). ;

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13C NMR (101 MHz, ) δ 167.57, 163.23, 154.27, 136.28, 129.74, 116.73, 99.05, 66.41, 44.38,
24.27.; GCMS: m/z 245
4.1.4.3. 4-methyl-6-morpholino-N-(thiophen-2-ylmethyl)pyrimidin-2-amine (5d)
1
White solid (82% yield); mp: 97ꢀ101°C; H NMR (400 MHz, CDCl3 ) δ 7.43 (d, J = 8.2 Hz
1H), 6.31 (t, J = 8.0 Hz 1H), 6.18 (d, J = 7.8 Hz 1H), 5.96 (s, 1H), 4.63 (bs, 1H), 4.11 (d, J =
8.1 Hz, 2H), 3.76 – 3.71 (m, 4H), 3.48 – 3.43 (m, 4H), 2.51 (s, 3H); 13C NMR (101 MHz,
CDCl3 ) δ 167.08, 162.12, 159.96, 149.84, 146.27, 120.35, 114.48, 97.73, 66.66, 51.55,
45.03, 25.47; GCMS: m/z 290
4.1.4.4. 2-(methyl(4-methyl-6-morpholinopyrimidin-2-yl)amino)ethanol (5e)
White solid (79% yield); mp: 58ꢀ62°C; 1H NMR (400 MHz, CDCl3 ) δ 6.14 (s, 1H) 3.83ꢀ3.75
(m, 6H), 3.70 (bs 1H) 3.62– 3.58 (m, 2H), 3.50 – 3.46 (m, 4H), 3.46 (s, 3H), 2.42 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ 167.11, 163.65, 161.75, 97.40, 66.36, 62.98, 53.56, 47.87, 37.69,
25.40; GCMS: m/z 252
4.1.4.5. 1-(4-methyl-6-morpholinopyrimidin-2-yl)piperidine-4-carbonitrile (5f)
1
White solid (80% yield); mp: 75ꢀ79°C; H NMR (400 MHz, ) δ 6.01 (s, 1H), 3.80ꢀ075 (m,
4H), 3.53 (t , J = 7.6 Hz, 4H), 3.16 – 3.12 (m, 2H), 2.90ꢀ2.86 (m, 2H), 2.59ꢀ 2.53 (m, 1H),
2.37 (s, 3H), 1.79ꢀ1.75 (m, 2H), 1.42ꢀ1.38 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 166.48,
163.93, 162.24, 120.02, 98.04, 66.05, 45.68, 44.91, 43.10, 29.06, 25.39; GCMS: m/z 287
4.1.4.6. 1-(4-methyl-6-morpholinopyrimidin-2-yl)piperidine-4-carboxamide (5g)
White solid (90% yield); mp: 187ꢀ191°C; 1H NMR (400 MHz, ) δ 7.24 (s, 1H), 6.74 (s, 1H),
5.91 (s, 1H), 4.56ꢀ 4.60 (m, 2H), 3.61 – 3.56 (m, 4H), 3.46 – 3.40 (m, 4H), 2.69 (td, J = 12.8,
2.1 Hz, 2H), 2.26 (tt, J = 11.6, 3.6 Hz, 1H), 2.08 (s, 3H), 1.65 (qd, J = 12.7, 2.2 Hz, 2H), 1.36
(qd, J = 12.5, 4.0 Hz, 2H). ; 13C NMR (101 MHz,) δ 177.07, 166.28, 163.54, 161.52, 91.84,
66.38, 44.37, 43.52, 42.62, 28.84, 24.69; GCMS: m/z 305
4.1.4.7. N-(3-methoxyphenethyl)-4-methyl-6-morpholinopyrimidin-2-amine (5h)
1
White solid (88% yield); mp: 96ꢀ100°C; H NMR (400 MHz, CDCl3 ) δ 7.29 (t, J = 7.7 Hz
1H), 6.89 (d, J = 7.9 Hz 1H), 6.80 (d, J = 8.0 Hz 1H), 6.71 (s, 1H), 5.91 (s, 1H) 4.65 (bs,
1H), 3.76 – 3.71 (m, 4H) 3.65 (s, 3H), 3.58ꢀ3.53 (m, 2H), 3.48 – 3.43 (m, 4H), 2.82 (t, J = 7.8
Hz, 2H), 2.46 (s, 3H); 13C NMR (101 MHz, CDCl3 ) δ 167.36, 164.71, 160.22, 159.80,

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141.48, 129.94, 121.61, 115.28, 112.86, 98.34, 66.36, 55.04, 48.25, 42.47, 35.79, 25.49;
GCMS: m/z 328
4.1.5. Synthesis of 1-(4-methyl-6-morpholinopyrimidin-2-yl)-1H-indole (5b)
To a 0 °C solution of indole (0.55g, 0.00469 mol) in 10 mL of DMF was added sodium
hydride (0.169g, 0.0070 mol, 60% dispersion in oil), stirred at 0°C for 30 min, then 5ꢀbromoꢀ
2ꢀchloroꢀ4ꢀmethylꢀ6ꢀmorpholinopyrimidine (3) (1g, 0.00469 mol) was added, and the
reaction mixture heated to 140°C for 2 h. The reaction mixture was cooled and diluted with
water, the solid separated was filtered and recrystallised from alcohol.
1
White solid (72% yield); mp: 176ꢀ180°C; H NMR (400 MHz, dmsoꢀd6) δ 8.48 (d, J = 8.0
Hz, 1H), 8.13 (s, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.31 (t, J = 8 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H),
6.79 (t, J = 8 Hz 1H), 6.18 (s, 1H), 3.75 (s, 4H), 3.61 (s, 4H), 2.56 (s, 3H); 13C NMR (101
MHz, dmsoꢀd6); 167.93, 164.04, 159.07, 143.65, 131.74, 129.05, 124.46, 122.68, 113.98,
112.79, 102.52, 99.13, 67.17, 49.82, 26.02; GCMS: m/z 294
4.2. In vitro Anticancer activity
4.2.1. Cell lines and culture conditions.
HeLa (cervix), NCIꢀH460 (lung), MCFꢀ7(breast), HepG2 (liver) and IMRꢀ32 (brain)
cell lines were purchased from National Centre for Cell Science, Pune, India. Cells were
cultured in MEM and RPMIꢀ1640 containing 10% FBS, 100 U of Penicillin G/ml and 100
µg/ml of streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.
4.2.2. Assessment of cell viability and morphology.
Effect of 4ꢀmethylꢀ6ꢀmorpholinopyrimidine derivatives on viability of HeLa and
NCIꢀH460 cells was assessed using 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyl tetrazolium
bromide (MTT) assay. Subꢀconfluent, HeLa, NCIꢀH460, MCFꢀ7, HepG2 and IMRꢀ32 were
seeded at 5 ×104 cells/ml in 96 well plates for 24 h. Cells were then treated with increasing
concentrations (5, 10 and 20 µM) of compounds for 24 h. DMSO treated cells were used as
the vehicle control. Following treatment, 10 µl of MTT (5 mg/ml) was added to each well
followed by lysis of the cells using 100 µl of 10% SDS in 0.01 N HCl and absorbance was
measured at 570 nm, with reference wavelength of 640 nm, using Tecan iꢀ200 microplate
reader. Percent viability was calculated by considering vehicle control as 100%. In another

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experiment, HeLa and NCIꢀH460 cells were treated as mentioned above and the cellular
morphological changes were photographed using phase contrast microscope.
4.2.3. Cell cycle analysis.
The distribution of HeLa and NCIꢀH460 cells in different phases of cellꢀcycle after
treatment with 4c and 5h compounds respectively was determined by flow cytometry.
Briefly, 0.1×106 cells were cultured in 12 well plate and treated with different concentration
(5, 10 and 20 µM) of compounds for 24 h. Post treatment cells were harvested, washed and
fixed in 70% chilled ethanol. Cells were then rehydrated in PBS (phosphate buffer saline) and
RNA was hydrolysed using 50 µl of Ribonuclease A (5 mg/ml in PBS, DNase free) for
overnight at room temperature. DNA was stained with Propidium Iodide (50 µM in PBS) for
30 min at 37oC and DNA content was analyzed on the FLꢀ2A channel of Flow Cytometer
(FACS Calibur, BD) equipped with a 488nm argon laser. The data was analyzed by Cell
Quest Pro software (BD) for the distribution of cells in different phases of cell cycle.
4.2.4. Detection of Apoptosis by Annexin-V/PI staining.
HeLa and NCIꢀH460 cells (0.1×106) cultured in 12 well plates were treated with or
without 4c and 5h compounds respectively for 24 h. cells were then harvested by gentle
trypsinization, washed with PBS and resuspended in binding buffer from BD Annexin–V kit.
Cells were then incubated with 5 µl FITC AnnexinꢀV and 5 µl of PI (Propidium Iodide) for 5
min at 37oC in the dark before analysis by flow cytometry (FACS Calibur, BD). The
percentages of cells positive for Annexin V, PI alone and both Annexin V and PI were
calculated by dot blot analysis using Cell Quest Pro software (BD Biosciences).
4.2.5. Analysis of mitochondrial membrane potential.
Alterations in mitochondrial membrane potential were determined using
cytofluorimetric, lipophilic cationic dye, 5,5’,6,6’ꢀtetrachloroꢀ1,1’,3,3’ꢀ tetraethylbenzimiꢀ
dazolylcarbocyanine iodide (JCꢀ1). HeLa and NCIꢀH460 cells (0.1×106) were treated with or
without 4c and 5h compounds respectively at different concentrations (5, 10 and 20 µM) for
24 h. Following treatment cells were harvested, washed with PBS and resuspend in 1 ml of
freshly prepared 2.5 µM JCꢀ1 solution followed by incubation at 370c for 20 min. Cells were
analyzed by flow cytometry (FACS Calibur, BD) with an excitation of 488 nm and emission
of 530 nm.

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4.2.6. Detection of intracellular Reactive Oxygen Species generation.
Intracellular ROS production was determined using cell permeable fluorescent probe
2, 7ꢀdichlorodihydro fluorescein diacetate (DCFDAꢀH2). Briefly, HeLa and NCIꢀH460 cells
were treated with 4c and 5h (10 µM) for 24h. After treatment, cells were washed with PBS
and stained with 15 µM DCFDAꢀH2 for 10 min at 30 °C. The relative fluorescence intensity
was analysed using fluorescence microscopy.
4.2.7. Determination of Caspase 3/7 activity.
Activation of Caspaseꢀ3/7 was measured in order to determine induction of apoptosis,
as per the manufacturer’s protocol supplied in caspaseꢀactivity detection kit (Promega). HeLa
and NCIꢀH460 cells (10 ×104 cells/ml) were cultured in 96 well plates and treatment was
carried out according to previously mentioned protocol for cell viability assay. Cell apoptosis
was then determined by measuring caspaseꢀ3/7 activity using Caspase Gloꢀ3/7 assay.
4.2.8. Nuclear morphological changes.
Changes in nuclear morphology of cells treated with compound 4c and 5h was
determined by staining with DAPI (4',6ꢀDiamidinoꢀ2ꢀPhenylindole). Briefly, cells were
treated with 4c and 5h compounds for 24 h, washed with PBS and fixed with 2% neutral
buffered formalin solution for 10 min. Cell permeabilization was carried out using 0.2%
tritonꢀx for 5min, followed by staining with DAPI (1 mg/mL) for 10 min. Cells were
examined for morphological changes under fluorescence microscope.
4.2.9. Statistical analysis.
Experiments were performed in multiple sets and results were analyzed for statistical significance,
using Oneꢀway ANOVA followed by Tukey’s multiple comparison tests utilizing GraphPad Prism5
software; Pꢀvalues are mentioned in figure legends.
4.3. DNA fragmentation assay.
The fragmentation of DNA after treatment with compounds was determined using
agarose gel electrophoresis. Briefly, HeLa and NCIꢀH460 cells were treated with and without
4c and 5h respectively at different concentrations (5, 10 and 20 µM) for 24 h. Cells were
harvested and total genomic DNA was isolated using standard protocol. The fragmentation of
DNA was determined by resolving DNA on 2% agarose gel. Standard DNA marker was used
to compare the fragmentation.

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4.4. Molecular modelling studies.
The modelling of the catalytic domain of the mTOR was done as described earlier [1].
Sequence of mTOR (IDP42345) was obtained from uniprot (www.uniprot.org), and as the
mTOR catalytic domain is localized between amino acids 2181 and 2516, the same was used
for homology modelling using the SwissꢀModel server (http://swissmodel. expasy.org/) using
the mTOR CryoꢀEM structure (5H64) available in PDB database (www.rcsb.org/). The
mTOR model was then prepared for docking using UCSF Chimera package [2], where
hydrogens where added and Gasteiger charges were computed. The small molecules, 4c and
5h were drawn in Marvin Sketch program (Chem Axon), and further processed with
Swissparam (www.swissparam.ch/), which provide topology and parameter for small organic
molecules. The docking analysis of the small molecules on the catalytic domain of the mTOR
was performed using Autodock Vina (Trott and Olson 2010) The docked structures were
analysed by DSV studio viewer (Accelrys Software Inc.) for determining the ligand
interactions.
Acknowledgements
The authors gratefully acknowledge the financial assistance from Department of
Science & Technology (DSTꢀSERB), Govt. of India, New Delhi under Major Research
Project No. SR/S1/OCꢀ58/2011 and University Grant Commission (UGC), New Delhi under
UPEꢀFARꢀI program, F. No. 14ꢀ3/2012 (NS/PE) and we are also grateful to the University
Scientific Instrumentation Centre (USIC), Karnatak University, Dharwad for Spectral
Analysis. One of the authors Supreet Gaonkar is thankful to UGC, New Delhi, for providing
Senior Research Fellowship (SRF).
Conflict of interest
None declared
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Figure Legends
Figure 1: Examples of PI3K/mTOR inhibitors.
Figure 2: ORTEP diagram of compound 4h with labeling showing 50% displacement
ellipsoids (Crystal structure drew in Mercury 3.7)
Figure 3:Packing diagram of 4h with Br…Br(3.590 Å) Br…π (3.491 Å) and also CH… π
(2.892 Å) interactions
Figure 4: Doseꢀdependent effect of 4c (A) and 5h (B) compounds on human cancer HeLa
and NCIꢀH460 cell proliferation. Results of MTT assay are represented as percentage of cell
viability at different concentrations of 4c and 5h, where error bars represents SEM of
triplicate determinations from three different assessments. ***p>0.001. The cellular
morphology of HeLa and NCIꢀH460 cells after treatment with or without 4c and 5h
compounds was analysed and photographed using phase contrast microscope (C).
Figure 5: Effect of 4c and 5h compounds on cell cycle progression of cancer cells.
Histograms represent distribution of HeLa and NCIꢀH460 cells in different phases of cell
cycle after treatment with or without 4c and 5h compounds, respectively at different
concentrations (0ꢀ20µM) (A). Bar graph indicates percentage of HeLa and NCIꢀH460 cell
population after treatment with or without 4c and 5h compounds, respectively in subG1, G1,
S, and G2–M phases of the cell cycle (B & C).

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Figure 6: Morpholino pyrimidine compounds induce cell apoptosis. Histograms represent
distribution of HeLa and NCIꢀH460 cells in different phases Q1 (necrotic/dead cells), Q2
(late apoptosis cells), Q3 (live cells) and Q4 (early apoptosis cells) after treatment with or
without 4c and 5h compounds, respectively at different concentrations (0ꢀ20µM) (A). Bar
graph indicates percentage of HeLa and NCIꢀH460 cell population in Q1, Q2, Q3 and Q4
phases after treatment with or without 4c and 5h compounds, respectively (B & C).
Figure 7: Loss of mitochondrial membrane potential induced by Morpholino
pyrimidine compounds. Density plot representing JCꢀ1ꢀstained HeLa and NCIꢀH460 cells
after treatment with different concentrations (0ꢀ20µM) of 4c and 5h compounds, respectively.
P1 represents population of cells with low mitochondrial membrane potential emitting green
fluorescence (A). Bar graph represents percentage of high mitochondrial membrane potential
cells emitting red fluorescent (P2) against low mitochondrial membrane potential cells
emitting green fluorescence (P1) (B & C).
Figure 8: Morpholino pyrimidine compounds induced apoptosis is associated with ROS
production, activation of caspases, chromatin condensation and DNA fragmentation.
Intracellular ROS production in HeLa and NCIꢀH460 cells after treatment with 4c and 5h
compounds respectively was determined by fluorescence microscopy using ROS specific
fluorescent probe (DCFDAꢀH2) (A). Activation of executioner caspases (caspase3/7) in
HeLa and NCIꢀH460 cells after treatment with 4c and 5h compounds respectively was
determined by measuring caspase 3/7 activity using caspase 3/7 Glo assay and represented as
fold increase in activity ***P>0.001 (B). Agarose gel profiles showing DNA fragmentation
in HeLa and NCIꢀH460 cells after treatment with 4c and 5h compounds respectively at
different concentrations (0ꢀ20µM) (C). DAPIꢀstaining showing the chromatinꢀcondensed
nuclei in apoptotic cells following treatment with 4c and 5h compounds. (D).‘‘M’’ is marker
Figure 9: Docking analysis of 4c and 5h shows the binding of these molecules at the
binding domain of the mTOR molecule. 4C at the binding domain of the mTOR shows
interactions with the neighbouring amino acids (A). Surface around the 4c molecule
interacting with the mTOR molecule (B). 5h at the binding domain of the mTOR showing
interactions with the neighbouring amino acids (C). Surface around the 5h molecule
interacting with the mTOR molecule (D).

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Figure 1: Examples of PI3K/mTOR inhibitors.
22x14mm (300 x 300 DPI)

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Figure 2: ORTEP diagram of compound 4h with labeling showing 50% displacement ellipsoids (Crystal
structure drew in Mercury 3.7)
18x12mm (300 x 300 DPI)

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Figure 3:Packing diagram of 4h with Br…Br(3.590 Å) Br…π (3.491 Å) and also CH… π (2.892 Å) interactions
18x14mm (300 x 300 DPI)

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Figure 4: Dose-dependent effect of 4c (A) and 5h (B) compounds on human cancer HeLa and NCI-H460 cell
proliferation. Results of MTT assay are represented as percentage of cell viability at different concentrations
of 4c and 5h, where error bars represents SEM of triplicate determinations from three different assessments.
***p>0.001. The cellular morphology of HeLa and NCI-H460 cells after treatment with or without 4c and 5h
compounds was analysed and photographed using phase contrast microscope (C).
24x23mm (300 x 300 DPI)

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Figure 5: Effect of 4c and 5h compounds on cell cycle progression of cancer cells. Histograms represent
distribution of HeLa and NCIꢀH460 cells in different phases of cell cycle after treatment with or without 4c
and 5h compounds, respectively at different concentrations (0ꢀ20µM) (A). Bar graph indicates percentage of
HeLa and NCIꢀH460 cell population after treatment with or without 4c and 5h compounds, respectively in
subG1, G1, S, and G2–M phases of the cell cycle (B & C).
27x25mm (300 x 300 DPI)

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Figure 6: Morpholino pyrimidine compounds induce cell apoptosis. Histograms represent distribution of HeLa
and NCIꢀH460 cells in different phases Q1 (necrotic/dead cells), Q2 (late apoptosis cells), Q3 (live cells) and
Q4 (early apoptosis cells) after treatment with or without 4c and 5h compounds, respectively at different
concentrations (0ꢀ20µM) (A). Bar graph indicates percentage of HeLa and NCIꢀH460 cell population in Q1,
Q2, Q3 and Q4 phases after treatment with or without 4c and 5h compounds, respectively (B & C).
27x26mm (300 x 300 DPI)