Synthesis, biological screening and molecular docking studies of novel 4,6-pyrimidine derivatives as EGFR-TK inhibitors

Article abstract of DOI:10.1007/s00044-016-1668-x

Novel 4, 6-disubstituted pyrimidine derivatives (5–16) were synthesized in four steps starting from 2,4-dichloropyrimidine and screened for their cytotoxicity using brine shrimp (Artemia Salina) lethality bioassay. The compounds such as 6, 11, 14 and 15 w

Full text of DOI:10.1007/s00044-016-1668-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1668-x
ORIGINAL RESEARCH
Synthesis, biological screening and molecular docking studies of novel
4,6-pyrimidine derivatives as EGFR-TK inhibitors
Siva Nagi Reddy Mule1 Sharmila Nurbhasha1 J.N. Kolla2 Surender Singh Jadav3
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Venkatesan Jayaprakash3 Lourdu Rani Bhavanam1 Hari Babu Bollikolla1
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Received: 16 October 2015 / Accepted: 4 July 2016
© Springer Science+Business Media New York 2016
Abstract Novel 4, 6-disubstituted pyrimidine derivatives
(5–16) were synthesized in four steps starting from 2,4-
dichloropyrimidine and screened for their cytotoxicity using
brine shrimp (Artemia Salina) lethality bioassay. The
compounds such as 6, 11, 14 and 15 were found to be more
toxic. The compounds were also studied for in vitro antic-
ancer properties using six different cancer cell lines viz
SIHA, PANC-1, MDA-MB-231, IMR-32, DU145 and
A549. The compound 14 was effective inhibitor of SIHA
and DU145, whereas compound 16 in Panc 1 and A549,
compound 7 in MDA-MB-231 and compound 6 in IMR 32
respectively. Molecular docking studies were carried out
using an X-ray crystallographic structure of epidermal
growth factor receptor tyrosine kinase to explore the pos-
sible mode of action of compounds as epidermal growth
factor receptor tyrosine kinase inhibitors.
Introduction
Pyrimidine derivatives were found to possess a wide range
of biological activities such as antifungal, anti-tubercular,
antibacterial (Ahluwalia and Madhu, 1996; Singh et al.,
2005; Shamroukh et al., 2005; Pasha et al., 2005), antiviral
(Lin et al., 1988; Holy et al., 2002; Rakesh et al., 2002;
Joule et al., 1995), anticancer (Skibo et al., 2002), anti-
oxidant (Vidal et al., 2000), and anti-inflammatory (Alam
et al., 2013). Four-substituted and 4,6-disubstituted pyr-
imidines bonded to a saturated heterocyclic unit at C-2
position of pyrimidine ring and related compounds have
attracted much attention as potent 5-HT2A receptor ligands,
including atypical antipsychotic, antidepressants, anxioly-
tics (Mokrosz et al., 1997; Glennon et al., 1997; Miyamoto
et al., 2000), dual EGFR/ErbB-2 kinase inhibitors (Li et al.,
2012), as potent HIV-1 NNRTIs (Yu et al., 2009), and
TRPV1 antagonists (Stec et al., 2008). Some of the antic-
ancer agents such as fluorouracil, gemcitabine, capecitabine
cytrabine, cladribine and bleomycin had pyrimidine ring
(Fig. 1).
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Keywords Suzuki coupling Boronic acids Pyrimidine
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derivatives Anticancer Molecular docking
Epidermal growth factor receptor tyrosine kinase
(EGFR-TK) is one of the important therapeutic targets in
human cancers. EGFR belongs to the ErbB family of TK
proteins which includes four members: ErbB1/HER1,
ErbB-2/Neu/HER2, ErbB-3/HER3 and ErbB-4/HER4
(Normanno et al., 2003). The participation of the EGFR
family of TKs in cancer proliferation suggests that an
inhibitor, which blocks the TK activity of the entire EGFR
family, could have substantial therapeutic potential
(Mendelsohn and Baselga, 2000). Gefitinib, erlotinib,
lapatinib, canceratinib, PD 153035, AG 1295, AG 1385,
etc. are examples of EGFR-TKIs, a biological target for
human cancer. Owing to the significant biological activities
of pyrimidine nucleus, we report herein the synthesis of
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1668-x) contains supplementary material,
which is available to authorized users.
* Hari Babu Bollikolla
dr.b.haribabu@gmail.com
1
Department of Chemistry, Acharya Nagarjuna University,
N. Nagar, Guntur, AP 522510, India
2
Department of Microbiology, Acharya Nagarjuna University,
N. Nagar, Guntur, AP 522510, India
3
Department of Pharmaceutical Sciences and Technology, Birla
Institute of Technology, Mesra, Ranchi, Jharkhand 835 215, India

Med Chem Res
F
OH
F
Fluorouracil
O
N
O
F
H2N
OH
N
HN
O
Gemcitabine
N
O
H
NH2
O
R
O
O
NH2
H
F
HN
N
NH2
R
O
R1
NH
N
N
S
O
N
O
OH
N
N
NH
N
N
N
H
N
N
HO
O
O
O
NH
H2N
HO
OH
O
Capecitabine
S
5-16
O
HN
HO
N
H
N
O
O
Bleomycin
OH
OH
O
NH2
NH
OH
OH
N
O
O
N
OH
HO
O
N
N
N
Cl
N
O
HO
HO
NH2
O
Cytrabine
Cladribine
HO
NH2
O
OH
Fig. 1 Reported structures of some anticancer agents having pyrimidine ring and the synthesized title compounds (5–16)
N
N
Cl
N
N
R
N
N
R
N
R
O
N
O
N
N
N
Cl
Cl
iii
N
iv
i
ii
N
N
O
NH
O
NH
NH
NH
2TFA salt
1
O
NH
R1
3a - d
2
4a - d
5-16
OH
B
F
Cl
OH
O
OH
B
OH
O
B
HO
O
OH
S
B
HO
O
N
S
OH
O
O
N
N
O
O
e
b
g
a
f
c
d
Scheme 1 Synthesis of 4,6-disubstituted pyrimidine derivatives (5–16). Reagents and conditions i 4-N-Boc-Amio piperidine, DIPEA, THF, rt, 4h;
ii a–d, Pd2(dba)3, X-Phos, DIPEA, RT, 4h; iii TFA, DCM, rt, 10 min; iv e–f, DIPEA, reflux, 10 min
some novel 4,6-disubstituted pyrimidine derivatives 5–16
(Scheme 1) derived starting from 2,4-dichloropyrimidine
and subsequently screened for their cytotoxicity properties.
Further, these compounds were evaluated for their antic-
ancer activity on six cancer cells, including SIHA (cervical
cancer), PANC-1 (pancreatic cancer), MDA-MB-231
(breast cancer), IMR 32 (neuroblastoma), DU 145 (prostate
cancer) and A549 (lung adenocarcinoma). Although several
EGFR-TKIs have been clinically validated for the treatment
of cancer patients, yet the search for new active molecules

Med Chem Res
Table 1 Synthesis of 4,6-pyrimidine derivatives (5–16)
N
NH
O
N
N
R
NH
R1
Compound
R
R1
Molecular formula
C30H29N5O4
M.P (°C)
Yield (%)
90
5
177–180
O
O
O
6
C28H26ClN5O2
111–112
95
Cl
O
7
8
C30H31N5O3
148–150
188–192
93
93
O
O
S
C26H25N5O3S
O
O
9
C24H22ClN5OS
236–238
94
Cl
S
10
11
C26H27N5O2S
C22H23N5O3S
206–209
188–191
93
94
O
O
S
S
O
12
C20H20ClN5OS
175–177
91
Cl
O
S
13
14
C22H25N5O2S
C25H26FN5O4
173–175
153–154
93
95
S
O
O
O
O
F
15
C23H23ClFN5O2
168–171
92
Cl
F

Med Chem Res
Table 1 continued
N
NH
O
N
N
R
NH
R1
Compound
R
R1
Molecular formula
C25H28FN5O3
M.P (°C)
Yield (%)
95
16
163–164
O
O
F
against EGFR-TK is still continuing. Hence we have
selected EGFR-TK as putative target for molecular docking
studies and explored the binding mode of our ligands with
active site of EGFR-TK.
corresponding piperidine nuclei. These data clearly confirm
the presence of specific functional groups present in final
synthesized compounds.
In vitro biological activity
Results and discussion
Chemistry
The compounds 5–16 (up to 300 μg/mL concentrations)
were tested for cytotoxicity using brine shrimp lethality
assay (Meyer et al., 1982) and compared with the activity of
podophyllotoxin (Table 2). The compounds 6, 11, 14 and
15 were showed ED50 values of 3.50, 3.69, 3.78 and
4.49 µg/mL, respectively and displayed equipotent
activity with respect to the standard Podophyllotoxin
(ED50 =3.69 µg/mL). The compounds 5 (ED50 =27.86 µg/mL),
compound 10 (ED50 = 39.49 µg/mL), compound 16
(ED50 = 39.64 µg/mL) were found to be exhibited moderate
cytotoxicity. The remaining analogs in the series were less
toxic.
Among the tested analogs, pyrimidine ring flanked by
4-phenoxyphenyl 6 (with R1=3-chlorophenyl on piperidine
ring), thiophene 11 (with R1=4-methylbenzoate on piperidine
ring) and 2-fluoro-4-methoxyphenyl 14 and 16 (with R1=4-
methylbenzoate and 3-chloro phenyl on piperidine ring
respectively) were exhibited cytotoxicity like Podophyllo-
toxin. The exact mechanism of cytotoxic activity between 4-
phenoxyphenyl (with R1=3-chlorophenyl on piperidine ring),
thiophene (with R1=4-methylbenzoate on piperidine ring)
and 2-fluoro-4-methoxyphenyl (with R1=4-methylbenzoate
and 3-chlorophenyl on piperidine ring) substitution on the
pyrimidine/piperidine ring was not established earlier, so
further we made an attempt to find out structural activity
relationship (SAR) with cytotoxicity and anticancer activities.
The compounds were further evaluated for anticancer
activity in six cancer cell lines (SIHA, PANC-1, MDA-MB-
231, IMR-32, DU145, and A549). Three response para-
meters such as growth inhibitory activity (GI50), total
growth inhibition (TGI), and lethal concentration (LC50)
were calculated for each cell line, using Doxorubicin and
The synthesis of 4,6-disubstituted pyrimidine derivatives
5–16 is shown in Scheme 1. The reaction of 2,4-dichlor-
opyrimidine (1) with tert-butylpiperidin-4-ylcarbamate in
the presence of diisopropylamine at room temperature
afforded pyrimidine chloride derivative 2. Suzuki coupling
of chloride intermediate 2 with various boronic acids (a–d)
was carried out at room temperature in presence of
Pd2(dba)3, X-Phos, diisopropylamine in tetrahydrofuran
(THF) to obtain corresponding compounds 3a–d in 85–95 %
yield. De-BOC of 3a–d was done by using trifluroacetic
acid (TFA) at 0 °C for 10 min to isolate compounds 4a–4d
as their respective TFA salts. The reaction of compounds
4a–d with isocyanates (e–f) in the presence of N,N-diiso-
propylethylamine (Hunig’s base) with reflux for 10 min,
resulted in urea derivatives 5–16 (Table 1). The yield of the
products varied from 90 to 95 %.
All the synthesized compounds were well characterized
1
by H & 13C-NMR, FT-IR and mass spectrometry. IR data
of compounds 5–16 showed a strong –C=O stretching band
around 1615–1657 cm−1 and a strong -NH stretching band
at 3295–3338 cm−1. All final compounds have strong
absorption around 3060–3435 cm−1 and around 1590 cm−1,
which are evidence for aromatic C–H and aromatic C–C
bonds respectively. All these compounds have shown a
singlet in the range of δ 8.5–8.9 ppm of corresponding
pyrimidine nuclei and another singlet in the range of δ 8.4–
8.6 ppm of corresponding urea linkage (–NH proton), and
signals for some protons in the range of δ 3.2–4.5 ppm,

Med Chem Res
Table 2 Brine shrimp lethality data of compounds 5–16
Compound
R
R1
ED50
(µg/mL)
5
27.86
O
O
O
O
O
6
3.50
Cl
7
8
42.46
40.52
O
O
O
S
S
9
43.79
Cl
10
11
39.49
3.69
O
S
S
S
S
O
O
12
44.23
Cl
13
14
43.28
3.78
O
O
O
O
F
15
16
4.49
Cl
O
O
F
39.64
3.69
O
F
Podophyllotoxin –
–

Med Chem Res
Fig. 2 The binding mode of
ligands (5–16) with the EGFR-
TK active site
Combretastatin as positive controls and data presented in
Table 3. Different concentrations of compounds were stu-
died for growth inhibition in cancer cell lines, and found to
be exhibited various levels of growth arrest vary from cell
line to cell line. The compound 14 showed better efficacy in
inhibiting cell proliferation of cervical cancer and prostate
cancer, followed by the compound 6 with GI50 range from
0.1 to 0.4 µM, the compound 14 required 1.3 µM for TGI in
DU145 but more than 100 µM in SIHA. The compound 16
was showed maximum growth inhibition in PANC 1 as well
A549 with GI50 of 0.47 and 0.11 µM, TGI of 1.6 and 4.3
µM respectively. The compound 7 was most effective
inhibitor of MDA-MB-231 with GI50 of 0.2 and TGI of 3.1
µM whereas compound 6 was highly potent inhibitor of
IMR 32 with GI50 of 0.65 and TGI of 4.6 µM correspond-
ingly. Even though positive controls such as Doxorubicin
and Combretastatin were highly potent growth inhibitors
than tested compounds but exhibited severe toxicity with
LC50 range of 1–5 µM. In the present study, all the com-
pounds from 5 to 16 were less toxic than Doxorubicin and
Combretastatin which might give a better therapeutic index
in the treatment of cancer, which is very promising for this
new group of compounds.
activity followed by 4-phenoxyphenyl and 2-benzthiazole
while R1 (substitution) with 4-methoxyphenyl showed
maximum activity followed by 4-methylbenzoate and 3-
chlorophenyl.
Molecular docking studies
EGFR-TKs are believed to have an essential role in the
instigation and the growth of the numerous cancers (Mostafa
et al., 2013). Moreover compounds bearing pyrimidine rings
were also reported as inhibitors of EGFR-TKs and few with
molecular docking to understand the key interactions (Mao
et al., 2013). Guided by these facts, we carried out the
molecular docking studies to explore the possible mode of
action of compounds 5–16 as EGFR-TKase inhibitors
(Fig. 2). The X-ray crystallographic structure of EGFR-TK
(PDB Code: 2J5F) was utilized for the current docking stu-
dies (Gangjee et al., 2013). The binding site of the co-crys-
tallized ligand (N-[4-(3-bromophenylamino) quinazolin-6-yl]
acrylamide) is well defined by the hydrophobic cavity lines
by Lys745, Glu762, Met766, Leu788, Thr790, Met793,
Cys797 and Thr854 (Blair et al., 2007; Ahsan et al., 2013).
All the molecules were docked against EGFR-TKase (PDB
Code: 2J5F) using XP-Glide-5.0 implemented in Maestro-
8.5. Docked poses of the molecules were carefully analyzed
SAR developed from the anticancer data showed that
pyrimidine derivatives having
R (substitution) with
2-fluoro-4-methoxyphenyl responsible for maximum

Med Chem Res
a
b
c
d
Fig. 3 3D Binding mode of compounds against EGFR-TK active site: a Compound 5 b. Compound 6 c Compound 7, d Compound 9
to understand the crucial interaction responsible for the
activity and found that the compound 5 and 6 were displayed
interactions with EGFR-TK. Both 5 and 6 were exhibited
hydrophobic interaction with Leu792 and Met793 and might
be crucial for anticancer activity. The other interactions of
compound 5 were as followed, H-bonding interaction with
Cys797 and a π–π interaction with Phe723 (Fig. 3a), and
compound 6, displayed two H-bonding interaction with
Lys875 (Fig. 3b). The compounds 7, 8 and 9 were
displayed hydrophobic interaction with Leu777 and Leu778
(Fig. 3c, d). The ATP binding pocket of EGFR-TK is con-
siderably hydrophobic, thus hydrophobic interactions plays
an important role in affinity toward EGFR-TK. Moreover it
was proposed that at least two hydrogen bonds are required
for small molecules to act as EGFR-TK inhibitor (Zuccotto et
al., 2010). In the present study, compounds 5, 6, 7, 8 and 9
might be showed hydrophobic interactions towards ATP
binding pocket to explored as EGFR-TK inhibitors. All the
other compounds were exhibited unusual interactions with
EGFR-TK and might be specific for other targets. Attempts
are in progress to find out SAR with therapeutic targets in
cancer.
Conclusion
A series of novel 4, 6-disubstituted pyrimidine derivatives
(5–16) were synthesized with good yields and were
screened for their in vitro cytotoxicity and anticancer
activity. The present study revealed that four compounds (5,
11, 14 and 15) were potent cytotoxic molecules in brine
shrimp lethality assay. The compounds were also studied
for in vitro anticancer properties using six different cancer
cell lines viz SIHA, PANC-1, MDA-MB-231, IMR-32,
DU145 and A549. The compound 14 was effective inhibitor
of SIHA and DU145, whereas compound 16 in Panc 1 and
A549, compound 7 in MDA-MB-231 and compound 6 in
IMR 32 respectively. Molecular docking studies were car-
ried out using an X-ray crystallographic structure of EGFR-
TK to explore the possible mode of action of compounds as
EGFR-TK inhibitors. Among potent anticancer compounds
in the present study, compound 6 was found to be exhibited
hydrophobic interaction with Leu792 and Met793 whereas
compound 7 was with Leu777 and 778. This binding pat-
tern with EGFR-TK might play a crucial role in its inhibi-
tion and anticancer activities. The present work provides

Med Chem Res
strong incentive for further development of 4,6-dis-
ubstituted pyrimidine derivatives as potential antitumor
agents for the treatment of metastatic cancer.
4 h. TLC indicated complete consumption of starting
material. The reaction mixture was diluted with water (5
mL) and extracted with ethyl acetate (15 mL), the combined
organic layer was washed with water (3 × 5 mL) followed
by brine solution. The organic layer was dried over anhy-
drous sodium sulphate, filtered and concentrated under
reduced pressure, to obtain the crude compounds. The crude
compounds were purified by column chromatography
(Silica gel: 100–200 mesh, elluant: 1:4 of ethyl acetate: n-
hexane) to isolate compounds 3a–d (Yield: 95, 90, 85, 88 %
respectively).
Materials and methods
All the chemicals and solvents used were purchased either
from Fluka or Merck. All the reagents were of analytical
grade. Thin layer chromatography (TLC) was performed on
the E. Merck AL silica gel 60 F254 plates and visualized
under UV light. The IR spectra were recorded on a Perkin
Elmer FT-IR spectrometer. The 1H NMR spectra were
recorded in CDCl3 on a Varian EM-360 spectrometer
operating at 400 MHz. The 13C NMR spectra recorded in
CDCl3 on a Varian EM-360 spectrometer operating at 100
MHz. The chemical shifts were reported in δ (ppm) using
TMS as an internal standard. The mass spectra were
recorded on Agilent ion trap MS. All the appropriate
boronic acids (a–d) and isocyanates (e–f) used for the
preparation of 5–16 were purchased from commercial
sources. All computational analysis were carried out on a
Dell Precision T3400n workstation with Intel core 2 quad
processor, 8 GB RAM, 500 GB hard disk, running on the
operating system Red Hat Enterprise 5.0 Linux (RHEL 5.0)
platform. Molecular docking simulations were carried out
using Protein preparation wizard, Ligprep and Glide module
implemented in Maestro-8.5 (Schrödinger LLC).
tert-Butyl 1-(6-(4-phenoxyphenyl)pyrimidin-4-yl)
piperidin-4-ylcarbamate (3a)
1H NMR (400 MHz, DMSO-d6): δ 1.33-1.28 (m, 2 H), 1.39
(s, 9H), 1.81 (d, 2H, J = 4.0 Hz), 3.07 (t, 2H, J = 5.0 Hz),
3.56 (brs, 2H), 4.48 (d, 2H, J = 12.0 Hz), 6.87 (d, 1H, J =
8.0 Hz), 7.10 (t, 4H, J = 2.0 Hz), 7.21 (t, 1H, J = 12.0 Hz),
7.28 (s, 1H), 7.45 (t, 2H, J = 4.0 Hz), 8.19 (d, 2H, J = 4.0
Hz), 8.53 (s, 1H); EI MS: m/z (rel. abund.%) 447.2 (M+,
100).
Isopropyl 1-(6-(benzo[b]thiophen-2-yl)pyrimidin-4-yl)
piperidin-4-ylcarbamate (3b)
1H NMR (400 MHz, DMSO-d6): δ 1.35–1.30 (m, 2 H), 1.39
(s, 9H), 1.84 (d, 2H, J = 3.0 Hz), 3.10 (t, 2H, J = 6.0 Hz),
3.59 (brs, 1H), 4.46 (d, 2H, J = 12.0 Hz), 6.89 (d, 1H, J =
8.0 Hz), 7.44–7.39 (m, 2H), 7.50 (s, 1H), 7.90–7.87 (m,
1H), 8.01–7.99 (m,1H), 8.40 (s, 1H),8.50 (s, 1H); EI MS:
m/z (rel. abund.%) 411.0 (M+, 100).
Synthesis of tert-butyl 1-(6-chloropyrimidin-4-yl)
piperidin-4-ylcarbamate (2)
To a solution of tert-butylpiperidin-4-ylcarbamate (4 g, 20
mmol) in THF (80 mL) was added N,N-diisopropylethyla-
mine (3.87 g, 30 mmol) followed by 2,4-dichloropyrimidine
(2.95 g, 20 mmol). The resulting mixture was stirred at
room temperature for 4 h. The reaction mixture was eva-
porated under reduced pressure and then water was added.
The resulted off-white solid was filtered and dried in
vacuum to afford compound 2 (Yield: 5.6 g, 92 %, off-white
solid); 1H NMR (400 MHz, DMSO-d6): δ 8.30 (s, 1H), 6.96
(s, 1H), 6.87 (d, 1H, J = 8.0 Hz), 4.27 (brs, 2H), 3.55 (brs,
1H), 3.08 (t, 2H, J = 7.0 Hz), 1.79 (d, 2H, J = 6.0 Hz), 1.38
(s, 9H), 1.33–1.23 (m, 2H). EI MS: m/z (rel. abund.%):
313.0 (M+, 100).
tert-Butyl 1-(6-(thiophen-2-yl)pyrimidin-4-yl)piperidin-
4-ylcarbamate (3c)
1H NMR (400 MHz, DMSO-d6): δ1.35-1.27 (m, 2H), 1.39
(s, 9H), 1.81 (d, 2H, J = 4.0 Hz), 3.07 (t, 2H, J = 6.0 Hz),
3.56 (brs, 1H), 4.42 (d, 2H, J = 6.0 Hz), 6.88 (d, 1H, J =
8.0 Hz), 7.20 (t, 1H, J = 8.0 Hz), 7.28 (s, 1H),7.69 (d, 1H, J
= 4.0 Hz), 8.02 (d, 1 H, J = 4.0 Hz), 8.41 (s, 1H); EI MS: m/z
(rel. abund.%) 361.2 (M+, 100).
tert-Butyl 1-(6-(2-fluoro-4-methoxyphenyl)pyrimidin-4-
yl)piperidin-4-ylcarbamate (3d)
General method for the synthesis of compounds (3a–d)
1H NMR (400 MHz, DMSO-d6): δ 1.35–1.27 (m, 2 H), 1.38
(s, 9H), 1.81 (d, 2H, J = 4.0 Hz), 3.09 (t, 2H, J = 7.0 Hz),
3.56 (brs, 1H), 3.79 (s, 3H), 4.34 (d, 2H, J = 6.0 Hz), 6.87
(d, 1H, J = 8.0 Hz), 7.07–7.03 (m, 1H),7.11 (s, 1H), 7.28 (t,
1H, J = 10.0 Hz), 7.42 (m,1H), 8.58 (s, 1H); EI MS: m/z
(rel. abund.%) 403.2 (M+, 100).
To a solution of compound 2 (1 g, 3.2 mmol) in THF (15
mL) was added sequentially corresponding boronic acid
(a–d, 4.8 mmol), DIPEA (6.4 mmol) followed by Pd2(dba)3
(10 mol%) and X-Phos (10 mol%) under argon. The resul-
tanting reaction mixture was stirred at room temperature for

Med Chem Res
General method for the synthesis of compounds (4a–d)
The crude compounds were purified by column chromato-
graphy (Silica gel: 100–200 mesh, elluant: 1:1, ethylacetate:
n-hexane) to obtain compounds 5–16 as off-white solids.
Yields of the products varied from 90 to 95 %.
A mixture of compound 3a–d (10 mmol), trifluroacetic acid
(5 vol) was stirred at 0 °C to room temperature for 10 min.
The reaction mixture was evaporated under vacuum and
dried to obtain the compounds, 4a–d as TFA salts and were
used in the next step without further purification.
1-(4-Methyl benzoate)-3-(1-(6-(4-phenoxyphenyl)
pyrimidin-4-yl)piperidin-4-yl)urea (5)
1
1-(6-(4-Phenoxyphenyl)pyrimidin-4-yl)piperidin-4-
amine (4a)
IR (KBr): νmax1651 (C=O), 3326 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.44–1.39 (m, 2H), 1.94 (d, 2H,
J = 4.5 Hz), 3.22–3.16 (m, 2H), 3.79 (s, 3H), 3.82–3.81 (m,
1H), 4.40 (brs, 2H), 6.40 (d, 1H, J = 6.0 Hz), 7.10 (d, 3H,
J = 2.0 Hz), 7.22 (t, 1H, J = 6.0 Hz), 7.30 (brs, 1H), 7.46 (t,
2H, J = 3.0 Hz), 7.52 (d, 2H, J = 4.5 Hz), 7.84 (d, 2H, J =
3.0 Hz), 8.18 (d, 2H, J = 3.0 Hz), 8.57–8.56 (m, 1H), 8.80
(s, 1H); 13C NMR (400 MHz, DMSO-d6 + TFA): δ 165.96,
161.65, 159.55, 158.89, 157.00, 155.84, 154.06, 145.13,
131.11, 130.37, 130.18, 128.92, 124.11, 121.64, 119.27,
118.02, 116.69, 97.94, 61.65, 46.32, 42.58, 31.52; EI MS:
m/z (rel. abund.%) 524.2 (M+, 100); analy. found for
C30H29N5O4–C 68.86, H 5.54, N 13.58, O 12.02 (%).
1H NMR (300 MHz, D2O): δ 1.76 (t, 2 H, J = 5.5 Hz),
2.23–2.20 (m, 3H), 3.24–3.16 (t, 1H, J = 12.0 Hz), 3.46 (t,
1H, J = 12.0 Hz), 3.68 (t, 1H, J = 14.0 Hz), 4.41 (d, 1H, J =
12.0 Hz), 5.16 (d, 1H, J = 18.0 Hz), 7.21 (t, 4H, J = 2.2 Hz),
7.32 (t, 1H, J = 7.5 Hz), 7.51 (t, 2H, J = 3.5Hz), 7.79 (d,
2H, J = 4.5 Hz), 8.61 (s, 1H); EI MS: m/z (rel. abund.%)
347.2 (M+, 100).
1-(6-(Benzo[b]thiophen-2-yl)pyrimidin-4-yl)piperidin-4-
amine (4b)
1H NMR (300 MHz, D2O): δ 1.75–1. 68 (m, 2H), 2.30 (d,
2H, J = 4.5 Hz), 3.32–3.28 (m, 2H), 3.67 (brs, 1H), 3.67
(brs, 1H), 4-45–4.43 (m, 2H), 7.04 (s, 1H), 7.58–7.49 (m,
2H), 8.01–8.98 (m, 2H), 8.07 (s, 1H), 8.52 (s, 1H); EI MS:
m/z (rel. abund.%) 311.2 (M+, 100).
1-(3-Chlorophenyl)-3-(1-(6-(4-phenoxyphenyl)
pyrimidin-4-yl)piperidin-4-yl)urea (6)
IR (KBr): νmax1657 (–C=O), 3331 (–NH) cm−1; H NMR
1
(400 MHz, DMSO-d6): δ 1.41–1.37 (m, 2H), 1.92 (d, 2H,
J = 3.0 Hz), 3.21 (t, 2H, J = 4.5 Hz), 3.83–3.80 (m, 1H),
4.42 (d, 2H, J = 4.5 Hz), 6.31 (d, 1H, J = 6.0 Hz), 6.93(d,
1H, J = 3.0 Hz), 7.10 (t, 4H, J = 1.5 Hz), 7.25–7.16 (m,
3H), 7.32 (s, 1H), 7.46 (t, 2H, J = 3.7 Hz), 7.67 (t, 1H, J =
1.5 Hz), 8.21 (d, 2 H, J = 3.0 Hz), 8.57 (d, 2H, J = 4.5Hz);
13C NMR (400 MHz, DMSO-d6): δ 161.88, 160.99 158.55,
158.02, 155.96, 154.23, 141.95, 133.06, 132.34, 130.19,
130.15, 128.71, 123.97, 120.58, 119.16, 117.99, 116.94,
115.97, 97.62, 46.42, 42.36, 31.52; EI MS: m/z (rel. abund.%)
500.1 (M+, 100); analy. found for C28H26ClN5O2–C 67.46,
H 5.04, N 14.20, O 6.20, Cl 7.10 (%).
1-(6-(Thiophen-2-yl)pyrimidin-4-yl)piperidin-4-amine
(4c)
1H NMR (300 MHz, D2O): δ 1.51–1.40 (m, 2H), 2.01 (d,
2H, J = 4.5 Hz), 3.10 (t, 2H, J = 6.0 Hz), 3.38 (brs, 1H),
4.55 (d, 2H, J = 3.0 Hz), 7.24 (t, 1H, J = 4.5 Hz), 7.34 (s,
1H), 7.77 (d, 1H, J = 3.0 Hz), 8.04 (d, 1H, J = 3.0 Hz), 8.51
(s, 2H); EI MS: m/z (rel. abund.%) 261.2 (M+, 100).
1-(6-(2-Fluoro-4-methoxyphenyl)pyrimidin-4-yl)
piperidin-4-amine (4d)
1-(4-Methoxybenzyl)-3-(1-(6-(4-phenoxyphenyl)
pyrimidin-4-yl)piperidin-4-yl)urea (7)
1H NMR (300 MHz, D2O): δ 1.78–1.63 (m, 2H), 2.25 (d,
2H, J = 6.0 Hz), 3.23 (t, 1H, J = 13.0 Hz), 3.40 (t, 1H, J =
6.0 Hz), 3.66–3.55 (m, 1H), 3.83 (s, 3H), 4.35 (d, 1H, J =
15.0 Hz), 5.15 (d, 1H, J = 6.0 Hz), 7.31–7.14 (m, 4.0 Hz),
8.62 (s, 1H); EI MS: m/z (rel. abund.%) 303.2.
1
IR (KBr): νmax1620 (–C=O), 3295 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.33–1.25 (m, 2H), 1.86 (d, 2H, J
= 4.0 Hz), 3.16 (t, 2H, J = 6.0 Hz), 3.73 (s, 3H), 3.74–3.73
(m, 1H), 4.13 (d, 2H, J = 2.0 Hz), 4.38 (d, 2H, J = 6.0 Hz),
5.95 (d, 1H, J = 8.0 Hz), 6.15 (t, 1H, J = 6Hz), 6.87 (d, 2H,
J = 4.0 Hz), 7.10 (t, 4H, J = 2.0 Hz), 7.21–7.15 (m, 3H),
7.27 (s, 1H), 7.45 (t, 2H, J = 4.0 Hz), 8.20 (d, 2H, J = 4.0
Hz), 8.53 (s, 1H); 13C NMR (400 MHz, DMSO-d6): ð
161.07, 160.76, 159.39, 159.01, 158.63, 158.49, 158.26,
157.87, 155.48, 152.06, 151.70, 132.93, 130.59, 130.19,
128.64, 125.03, 124.72, 120.10, 118.35, 117.11, 114.22,
General method for the synthesis of compounds (5–16)
To a stirred mixture of compounds 4a–4d (10 mmol) in
N,N-diisopropylethylamine (10 Vol) was added appropriate
isocyanates (e–f, 10 mmol) and refluxed for 10 min. The
reaction mixture was evaporated under reduced pressure
and diluted with water to obtain crude compounds (5–16).

Med Chem Res
113.90, 99.49, 55.16, 46.13, 42.63, 32.32; EI MS: m/z (rel.
abund.%) 510.1 (M+, 100); analy. found for C30H31N5O3–C
70.60, H 6.11, N 13.79, O 9.50 (%).
C26H27N5O2S–C 65.91, H 5.78, N 14.80, O 6.72, S 6.80
(%).
1-(4-Methyl benzoate)-3-(1-(6-(thiophen-2-yl)pyrimidin-
4-yl)piperidin-4-yl)urea (11)
1-(4-Methyl benzoate)-3-(1-(6-(benzo[b]thiophen-2-yl)
pyrimidin-4-yl)piperidin-4-yl) urea (8)
1
IR (KBr): νmax1650 (–C=O), 3316 (–NH) cm−1; H NMR
1
IR (KBr): νmax1641 (–C=O), 3310 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.43–1.35 (m, 2H), 1.94 (d, 2 H, J
= 6.0 Hz), 3.21 (t, 2H, J = 6.0 Hz), 3.80 (s, 3H), 3.83–3.82
(m, 1H), 4.39 (d, 2H, J = 6.0 Hz), 6.39 (d, 1H, J = 4.0 Hz),
7.20–7.18 (t, 1H, J = 8.0 Hz), 7.31 (s, 1H), 7.52 (d, 2H, J =
4.0 Hz), 7.70–7.69 (d, 1H, J = 4.0 Hz), 7.84 (d, 2H, J = 4.0
(400 MHz, DMSO-d6): δ 1.45–1.37 (m, 2H), 1.95 (d, 2H, J
= 4.0 Hz), 3.26 (t , 2H, J = 6.0 Hz), 3.79 (s, 3H), 3.85–3.83
(m, 1H), 4.43–4.40 (d, 2H, J = 3.0 Hz), 6.40–6.38 (d, 1H, J
= 8.0 Hz), 7.43–7.40 (m, 2H), 7.54–7.50 (t, 2H, J = 4.0 Hz),
7.84–7.82 (d, 2H, J = 4.0 Hz), 7.90–7.88 (m, 1H), 8.01–
7.99 (m, 1H), 8.42 (s, 1H), 8.52 (s, 1H), 8.81 (s, 1H); 13C
NMR (400 MHz, DMSO-d6): δ 165.49, 161.41, 158.07,
156.84, 154.03, 145.08, 143.33, 140.06, 139.89, 130.33,
125.57, 124.76, 124.34, 123.44, 122.69, 121.66, 116.69,
97.18, 51.65, 46.40, 42.41, 31.50; EI MS: m/z (rel. abund.%)
488.1 (M+, 100); analy. found for C26H25N5O3S–C 64.15,
H 5.06, N 14.37, O 9.88, S 6.54 (%).
Hz), 8.04 (d, 1H, J = 4.0 Hz), 8.44 (s, 1H), 8.90 (s, 1H); 13
C
NMR (400 MHz, DMSO-d6): δ 165.96, 161.54, 158.03,
156.99, 154.02, 145.08, 143.22, 130.33, 129.37, 128.34,
126.78, 121.67, 116.71, 95.95, 51.65, 46.41, 42.33, 31.51.
EI MS: m/z (rel. abund.%) 438.1 (M+, 100); analy found
for C22H23N5O3S - C 60.36, H 5.33, N 15.98, O 10.96,
S 7.37 (%).
1-(1-(6-(Benzo[b]thiophen-2-yl)pyrimidin-4-yl)
piperidin-4-yl)-3-(3-chlorophenyl)urea (9)
1-(3-Chlorophenyl)-3-(1-(6-(thiophen-2-yl)pyrimidin-4-
yl)piperidin-4-yl)urea (12)
IR (KBr): νmax1639 (–C=O), 33140 (–NH) cm−1; 1H NMR
(400 MHz, DMSO-d6): δ 1.44–1.36 (m, 2H), 1.95 (d, 2H, J
= 6.0 Hz), 3.24 (t, 2H, 6.0 Hz), 3.83–3.82 (m, 1H), 4.43 (d,
2H, J = 4.0 Hz), 6.32 (d, 1H, J = 8.0 Hz), 6.94 (d, 1H, J =
8.0 Hz), 7.25–7.16 (m, 2H), 7.43–7.40 (m, 2H), 7.54 (s,
1H), 7.67 (s, 1H), 7.90–7.88 (m,1H), 8.01–7.99 (m, 1H),
8.41 (s, 1H), 8.52 (s, 1H), 8.58 (s, 1H); 13C NMR (400
MHz, DMSO-d6): δ 161.44, 158.15, 156.97, 154.26,
143.49, 141.98, 140.07, 139.90, 133.09, 130.2, 125.54,
124.74, 124.34, 123.37, 122.67, 120.60, 116.97, 115.99,
97.15, 46.41, 42.42, 31.56; EI MS: m/z (rel. abund.%) 464.1
(M+, 100); analy. found for C24H22ClN5OS–C 62.14; H
4.76; N 15.11; O 3.43; S 3.93; Cl 7.63 (%).
1
IR (KBr): νmax1633 (–C=O), 3296 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.41–1.34 (m, 2H), 1.93 (d, 2H, J
= 6.0 Hz), 3.20 (t, 2H, J = 6.0 Hz), 3.81 (brs, 1H), 4.39 (d,
2H, J = 6.0 Hz), 6.31 (d, 1H, J = 8.0 Hz), 6.94 (d, 1H, J =
8.0 Hz), 7.25–7.16 (m, 3H), 7.31 (s, 1H), 7.70–7.66 (m,
3H), 8.03 (d, 1H, J = 4.0 Hz), 8.43 (s, 1H), 8.58 (s, 1H); 13
C
NMR (400 MHz, DMSO-d6): δ 161.62, 158.10, 157.06,
154.35, 143.24, 142.00, 133.16, 130.30, 129.46, 128.44,
126.86, 120.73, 117.05, 116.09, 96.02, 46.47, 42.46, 31.61;
EI MS: m/z (rel. abund.%) 414.1 (M+, 100); analy. found
for C20H20ClN5OS–C 58.05, H 4.87, N 16.90, O 3.89, S
7.74, Cl 8.55 (%).
1-(4-Methoxybenzyl)-3-(1-(6-(benzo[b]thiophen-2-yl)
pyrimidin-4-yl)piperidin-4-yl)urea (10)
1-(4-Methoxybenzyl)-3-(1-(6-(thiophen-2-yl)pyrimidin-
4-yl)piperidin-4-yl)urea (13)
1
1
IR (KBr): νmax1625 (–C=O), 3293 (–NH) cm−1; H NMR
IR (KBr): νmax1622 (–C=O), 3295 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.35–1.27 (m, 2H), 1.89 (d, 2H, J
= 3.0 Hz), 3.20 (t, 2H, J = 6.0 Hz), 3.72 (s, 3H), 3.74 (m,
1H), 4.14 (d, 1H, J = 4.0 Hz), 4.40 (d, 2H, J = 6.0 Hz), 5.97
(d, 1H, J = 8.0 Hz), 6.17 (t, 1H, J = 6.0 Hz), 6.88 (d, 2H, J
= 4.0 Hz), 7.18 (d, 2H, J = 4.0 Hz),7.42 (d, 2H, J = 4.0 Hz),
7.51 (s, 1H), 7.89–7.87 (m, 2H), 8.01–7.99 (m, 1H), 8.40 (s,
1H), 8.50 (s, 1H); 13C NMR (400 MHz, DMSO-d6): δ
161.50, 158.22, 158.11, 157.40, 156.99, 143.50, 140.11,
139.96, 132.77, 128.40, 125.63, 124.83, 124.42, 123.42,
122.75, 113.67, 97.19, 55.07, 46.50, 42.59, 32.00; EI MS:
m/z (rel. abund.%) 474.1 (M+, 100); analy. found for
(400 MHz, DMSO-d6): δ 1.32–1.24 (m, 2H), 1.86 (d, 2H, J
= 4.0 Hz), 3.15 (t, 2H, J = 5.0 Hz), 3.72 (s, 3H), 3.73 (brs,
1H), 4.13(d, 2H, J = 2.0 Hz), 4.36 (d, 2H, J = 6.0 Hz), 5.95
(d, 1H, J = 8.0 Hz), 6.15 (t, 1H, J = 6.0 Hz), 6.88–6.85 (d,
2H, J = 6.0 Hz), 7.20–7.15 (m, 3H), 7.29 (s, 1H), 7.69 (d,
1H, J = 4.0 Hz), 8.03 (d, 1H, J = 4.0Hz), 8.42 (s, 1H); 13C
NMR (400 MHz, DMSO-d6): δ 161.55, 158.03, 158.00,
157.27, 156.94, 143.21, 132.73, 129.35, 128.31, 126.75,
113.59, 95.92, 55.00, 46.42, 42.46, 31.93. EI MS: m/z
(rel. abund.%) 424.1 (M+, 100); analy. found for
C22H25N5O2S–C 62.31, H 2.01, N 16.54, O 7.57, S 7.60 (%).

Med Chem Res
1-(4-Methylbenzoate)-3-(1-(6-(2-fluoro-4-
Brine shrimp lethality assay
methoxyphenyl)pyrimidin-4-yl)piperidin-4-yl)urea (14)
Brine shrimp (Artemia Salina) nauplii were hatched using
brine shrimp eggs in a conical shaped vessel (1 L), filled
with sterile, artificial sea water of 38 g L−1 of sea salt and
adjusted to pH 8.5 using 1 N NaOH and kept under constant
aeration for 48 h. After hatching, ten nauplii were drawn
through a pipette and placed in each vial containing 4.5 mL
brine solution and added 300 µg mL−1 concentration of
compounds and the final volume was made up to 5 mL
using brine solution and maintained 37 °C for 24 h under
the light of incandescent lamps. Assays were carried out in
duplicates. The percentage lethality was determined by
comparing the mean surviving larvae of test and control
tubes. Podophyllotoxin was used as a positive control.
1
IR (KBr): νmax1582 (–C=O), 3338 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.43–1.36 (m, 2H), 1.94 (d, 2H, J
= 6.0 Hz), 3.22 (t, 2H, J = 5.0 Hz), 3.79 (s, 3H), 3.91–3.90
(m, 1H), 4.31 (d, 2H, J = 4.0 Hz), 6.41 (d, 1H, J = 8.0 Hz),
7.07–7.04 (m, 1H), 7.15 (s, 1H), 7.29 (t, 1H, J = 10 Hz),
7.43–7.41 (m, 1H), 7.52 (d, 2H, J = 4.0 Hz), 7.84 (d, 2 H, J
= 4.0 Hz), 8.60 (s, 1H), 8.81 (s, 1H); 13C NMR (400 MHz,
DMSO-d6): δ 166.14, 160.36, 159.30, 158.80, 158.29,
157.79, 155.82, 155.03, 154.28, 151.79, 151.58, 148.18,
145.23, 130.47, 121.86, 119.48, 117.38, 117.29, 116.77,
115.60, 113.46, 103.39, 56.08, 51.70, 45.69, 31.68;
EI MS: m/z (rel. abund.%) 480.1 (M+, 100); analy. found
for C25H26FN5O4–C 62.63, H 5.48, N 14.61, O 13.36,
F 3.95 (%).
In vitro anticancer activity
The cancer cell lines, SIHA, PANC 1, MDA MB 231, IMR
32, DU 145 and A549 used in this study were procured
from the American Type Culture Collection (ATCC), USA.
The synthesized test compounds were evaluated for their
in vitro antiproliferative activity on these six different
human cancer cell lines. All the cell lines were grown in
Dulbecco’s modified Eagle’s medium (containing 10 %
FBS in a humidified atmosphere of 5 % CO2 at 37 °C). Cells
were trypsinized when sub-confluent and seeded in 96-well
plates in 100 μL aliquots at plating densities depending on
the doubling time of individual cell lines. A protocol of 48 h
continuous drug exposure was used, and a sulforhodamine
B (SRB) cell proliferation assay was used to estimate cell
viability or growth. 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 and were
incubated for 48 h with different doses (0.01, 0.1, 1, 10,
100 µM) of prepared derivatives. After 48 h incubation at
37 °C, cell mono layers were fixed by the addition of 10 %
(wt/vol) cold trichloroacetic acid and incubated at 4 °C for
1 h and were then stained with 0.057 % SRB dissolved in 1 %
acetic acid for 30 min at room temperature. Unbound SRB
was washed with 1 % acetic acid. The protein bound dye
was dissolved in 10 mM tris base solution for OD deter-
mination at 510 nm using a microplate reader (Enspire,
Perkin Elmer, USA). Using the seven absorbance mea-
surements [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 concentration levels. Percentage growth inhibition
was calculated as:
1-(3-Chlorophenyl)-3-(1-(6-(2-fluoro-4-methoxyphenyl)
pyrimidin-4-yl)piperidin-4-yl) urea (15)
1
IR (KBr): νmax1634 (–C=O), 3302 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.43–1.35 (m, 2H), 1.93 (d, 2H, J
= 6.0 Hz), 3.22 (t, 2H, J = 5.0 Hz), 3.80 (s, 3H), 3.82–3.81
(m, 1H), 4.32 (d, 2H, J = 6.0 Hz), 6.32 (d, 1H, J = 8.0 Hz),
6.94–6.91 (m, 1H), 7.09–7.05 (m, 1H), 7.30–7.16 (m, 4H),
7.42–7.27 (m, 1H), 8.61 (s, 1H), 7.66–7.65 (m, 1H), 8.57
(s,1H); 13C NMR (400 MHz, DMSO-d6): ð 161.28, 157.73,
155.92, 155.44, 154.23, 152.70, 141.95, 133.05, 130.19,
126.13, 120.58, 117.30, 116.98, 115.97, 114.47, 102.54,
102.42, 55.68, 46.27, 42.45, 31.42; EI MS: m/z (rel. abund.
%) 456.1 (M+, 100); analy. found for C23H23ClFN5O2 C
60.60, H 5.07, F 4.21, N 15.33, O 7.05, Cl 7.74 (%).
1-(4-Methoxybenzyl)-3-(1-(6-(2-fluoro-4-
methoxyphenyl)pyrimidin-4-yl)piperidin-4-yl) urea (16)
1
IR (KBr): νmax1615 (–C=O), 3319 (–NH) cm−1; H NMR
(400 MHz, DMSO-d6): δ 1.34–1.23 (m, 2H), 1.86 (d, 2H, J
= 4.0 Hz), 3.17 (t, 2H, J = 5.0 Hz), 3.75 (s, 4H), 3.79 (s,
3H), 4.13 (d, 2H, J = 4.0 Hz), 4.29 (d, 2H, J = 6.0 Hz), 5.95
(d, 1H, J = 8.0 Hz), 6.15 (t, 1H, J = 6.0 Hz), 6.88 (d, 2H, J
= 6.0 Hz), 7.08–7.04 (m, 1H), 7.17 (t, 3H, J = 2.5 Hz),
7.29–7.24 (dd, 1H, J = 7.0, 3.8 Hz), 7.42–7.40 (m, 1H),
8.59 (s, 1H); 13C NMR (400 MHz, DMSO-d6):
δ
161.38, 158.03, 157.29, 155.96, 155.88, 154.95, 153.51,
133.76, 128.67, 117.48, 117.24, 116.51, 116.43, 115.43,
113.59,101.62, 55.00, 53.51, 46.40, 42.28, 41.76, 31.84; EI
MS: m/z (rel. abund.%) 466.1 (M+, 100); analy. found
for C25H28FN5O3–C 64.48, H 6.07, N 15.02, O 10.33,
F 4.09 (%).
ꢀ
ꢁ
ðTi ꢀ TzÞ
ðC ꢀ TzÞ
´ 100 for concentrations for which Ti>= ¼ Tz

Med Chem Res
ꢀ
ꢁ
References
ðTi ꢀ TzÞ
´ 100 for concentrations for which Ti < Tz:
Tz
Ahluwalia VK, Madhu B (1996) A facile one pot synthesis of some
new derivatives of pyrano [2,3-d] pyrimidines as potential anti-
bacterial and antifungal agents. Indian J Chem 35B:742–744
Ahsan MJ, Khalilullah H, Yasmin S, Jadav SS, Govindasamy J (2013)
Synthesis, characterisation, and in vitro anticancer activity of
curcumin analogues bearing pyrazole/pyrimidine ring targeting
EGFR tyrosine kinase. Biomed Res Int 2013:239354
Three dose response parameters were calculated for each
experimental agent (Boyd and Paul, 1995). Growth inhibi-
tion of 50 % (GI50) was calculated from [(Ti−Tz)/(C−Tz)] ×
100 = 50, which is the drug concentration resulting in a 50 %
reduction in the net growth increase (as measured by SRB
staining) in control cells during the drug incubation. The
drug concentration resulting in total growth inhibition (TGI)
was calculated from Ti = Tz. The LC50 (concentration of
the drug resulting in a 50 % reduction in cell growth at the
end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells (toxicity) following
treatment was calculated from [(Ti − Tz)/Tz] × 100 = −50.
Values were calculated for each of these three parameters if
the level of activity is reached; however, if the effect is not
reached or is exceeded, the value for that parameter was
expressed as greater or less than the maximum or minimum
concentration tested. Standard error ( ) values were also
presented in the Table 3.
Alam MJ, Ahsan MJ, Alam O, Khan SA (2013) Synthesis of 4-(5-
chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-(substituted phe-
nyl) pyrimidin-2-ol analogues as
8 anti-inflammatory and
analgesic agents. Lett Drug Des Discov 10:776–782
Blair JA, Rauh D, Kung C, Yun CH, Fan QW (2007) Structure-guided
development of affinity probes for tyrosine kinases using che-
mical genetics. Nat Chem Biol 3:229–38
Boyd MR, Paul KD (1995) Some practical considerations and appli-
cations of the drug discovery screen. Drug Dev Res 34:91–109
Gangjee A, Namjoshi OA, Yu J, Ihnat MA, Thorpe JE, Bailey-Downs
LC (2013) N2-Trimethylacetyl substituted and unsubstituted-N4-
phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyr-
imidine-2,4-diamines: design, cellular receptor tyrosine kinase
inhibitory activities and in vivo evaluation as antiangiogenic,
antimetastatic and antitumor agents. Bioorg Med Chem 21:1312–
1323
Glennon RA, Young R, Dukat M, Cheng Y (1997) Initial character-
ization of PMMA as a discriminative stimulus. Pharmacol Bio-
chem Behav 57:151–8
Holy A, Votruba I, Masojidkova M, Andrei G, Snoeca R, Naesens L et
al. (2002) 6-[2-(Phosphonomethoxy) alkoxy] pyrimidines with
antiviral activity. J Med Chem 45:1918–1929
Joule JA, Mills K, Smith GF (1995) Heterocyclic chemistry, 3rd ed.
CRC Press, London, 189–194. editors
Molecular docking
Molecular docking simulations were carried out using
Maestro-8.5 (Schrodinger LLC) installed on RHEL-5
operating system. The structures of molecules were sket-
ched using build module implemented in Maestro-8.5. All
the sketched molecules were prepared for docking
using Ligprep module. The X-ray crystal structure of
EGFR-TKase (PDB Code: 2J5F) was downloaded from
protein data bank (http://www.rcsb.org/pdb/explore.do?
structureId=2j5f). The protein was prepared for docking
using the Protein Preparation Wizard, keeping OPLS2001
Force field for minimization. The grid was constructed,
keeping the reference ligand (34-JAB) as the center of the
grid using GLIDE (software v5.5) Receptor Grid Genera-
tion module. The ligands were then docked with the
receptor grid generated using XP-Docking implemented in
Glide-5. The docked conformers were analyzed through
XP-Visualizer.
Li S, Guo C, Zhao H, Tang Y, Lan M (2012) Synthesis and biological
evaluation of 4-[3-chloro-4-(3-fluorobenzyloxy) anilino]-6-(3-
substituted-phenoxy)pyrimidines as dual EGFR/ErbB-2 kinase
inhibitors. Bioorg Med Chem 20:877–885
Lin TS, Guo JY, Schinazi RF, Chu CK, Xiang JN, Prussof WH (1988)
Synthesis and antiviral activity of various 3’-azido analogs of
pyrimidine deoxyribonucleosides against human immunodefi-
ciency virus (HIV-I, HTLV-III/LAV). J Med Chem 31:336–340
Mao Y, Zhu W, Kong X, Wang Z, Xie H, Ding J (2013) Design,
synthesis and biological evaluation of novel pyrimidine, 3-cya-
nopyridine and m-amino-N-phenylbenzamide based monocyclic
EGFR tyrosine kinase inhibitors. Bioorg Med Chem 21:3090–
3104
Mendelsohn J, Baselga J (2000) The EGF receptor family as targets for
cancer therapy. Oncogene 19:6550–6565
Meyer BN, Ferrigni NR, Putnam JE, et al. (1982) Brine shrimp: a
convenient general bioassay for active plant constituents. Planta
Med 45:31–34
Miyamoto S, Duncan GE, Mailman RB, Lieberman JA (2000)
Developing novel antipsychotic drugs: strategies and goals. . Curr
Opin CPNS Invest Drugs 2:25–39
Mokrosz MJ, Duszynska B, Klodzinska A, Deren-Wesolek A, Choj-
nackaWojcik E, Baranowski TC, Abdou IM, Redmore NP,
Acknowledgment The authors are thankful to Laila Impex Research
& Development Center, Vijayawada, A.P. India and CSIR- IICT,
Hyderabad for their help in study of biological activities of the com-
pounds. Also thankful to Mohamed Jawed Ahsan, Department of
Pharmaceutical Chemistry, Maharishi Arvind College of Pharmacy,
Jaipur, Rajasthan, India for his helpful discussions. Thanks to CSIR-
New Delhi, for support to chemicals and reagents (02(198)/EMR-II).
Strekowski
L (1997) 4-(3-furyl)-2-(4-methylpiperazino) pyr-
imidines: potent 5-HT2A receptor antagonists. Bioorg Med Chem
Lett 7:1635–1638
Mostafa HM, Dalal A, El-Ella A (2013) Quinazoline and tetra-
hydropyridothieno[2,3-d]pyrimidine derivatives as irreversible
EGFR tyrosine kinase inhibitors: influence of the position 4
substituent. Med Chem Comm 4:1202–1207
Compliance with ethical standards
Normanno N, Maiello MR, De Luca A (2003) Epidermal growth
factor receptor tyrosine kinase inhibitors (EGFR-TKIs): simple
Conflict of interest The authors declare that they have no com-
peting interests.

Med Chem Res
drugs with a complex mechanism of action? J Cell Physiol
topoisomerase II to DNA cross-linkers with activity against
protein kinases. J Med Chem 45:5543–5555
194:13–19
Pasha TY, Udupi RH, Bhat AR (2005) Synthesis and antimicrobial
screening of some pyrimidine derivatives. Indian J Heterocycl
Chem 15:149–152
Stec MM, Bo Y, Chakrabarti PP, Liao L, Ncube M, et al. (2008)
Substituted aryl pyrimidines as potent and soluble TRPV1
antagonists. Bioorg Med Chem Lett 18:5118–5122
Vidal A, Ferrandiz ML, Ubeda A, Guillen I, Riguera R, Quintela JM,
et al. (2000) Effects ofsome isoxazolpyrimidine derivatives
on nitric oxide and eicosanoid biosynthesis. Life Sci 66:
125–131
Yu M, Liu X, Li Z, Liu S (2009) Synthesis and biological evaluation
of novel 2-(substituted phenylaminocarbonylmethylthio)-6-(2,6-
dichlorobenzyl)-pyrimidin-4(3H)-ones as potent HIV-1 NNRTIs.
Bioorg Med Chem 17:7749–7754
Rakesh K, Nath M, Tyrell DL (2002) Design and synthesis of novel 5-
substituted acyclic pyrimidine nucleosides as potent and selective
inhibitors of hepatitis B virus. J Med Chem 45:2032–2040
Shamroukh AH, Rashed AE, Sayed HH (2005) Synthesis of some
pyrazolo [3,4] pyrimidine derivatives for biological evaluation.
Phosphorus Sulphur Silicon Relat Elem 180:2347–2360
Singh DV, Misha AR, Misha RM, Pandey AK, Dwivedi AK (2005)
Synthesis and fungicidal activity of benzofuran incorporated
substituted pyrimidines. Indian J Hetrocycl Chem 14:319–322
Skibo EB, Huang X, Martinez R, Lemus RH, Craigo WA, Derr RT
(2002) Pyrimidoquinazoline-based antitumor agents: Design of
Zuccotto F, Ardini E, Casale E, Angiolini M (2010) Through the
“Gatekeeper Door”: exploiting the active kinase conformation.
J Med Chem 53:2681–269