Design and synthesis of quinazolinone tagged acridones as cytotoxic agents and their effects on EGFR tyrosine kinase

Article abstract of DOI:10.1002/ardp.201400065

In a quest for finding potent cytotoxic molecules, we have designed and synthesized a new scaffold by tagging quinazolinones with an acridone moiety. The new acridone-4-carboximide derivatives were evaluated for their cytotoxic potentials against the MCF7 breast cancer cell line and three colon cancer cell lines (LS174T, SW1398, and WiDr). Compound 26 showed relatively potent cytotoxic activity among the derivatives, against all the cell lines tested. Mechanistic studies for the selected derivatives 7, 8, 16, 17, 25, and 26 were conducted through in vitro EGFR tyrosine kinase inhibition studies. The results indicate that compound 26 has a better EGFR tyrosine kinase inhibitory profile. The in vitro EGFR inhibition data was correlated with the cytotoxic properties, and molecular docking studies were performed with regard to the receptor autophosphorylation sites of the protein kinase domain of the EGFR.

Full text of DOI:10.1002/ardp.201400065

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
1
Full Paper
Design and Synthesis of Quinazolinone Tagged Acridones as
Cytotoxic Agents and Their Effects on EGFR Tyrosine Kinase
Yarlagadda Rajesh Babu1, Mantripragada Bhagavanraju1, Gade Deepak Reddy2,
Godefridus J. Peters4, and Velivela V. S. Rajendra Prasad3,4
1
Department of Pharmaceutical Chemistry, Gland Institute of Pharmaceutical Sciences, Narsapur, India
Medicinal Chemistry Research Division, Vishnu Institute of Pharmaceutical Education and Research,
2
Narsapur, India
3
Department of Pharmaceutical Chemistry, Sitha Institute of Pharmaceutical Sciences, Bachupally, Hyderabad,
India
4
Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands
In a quest for finding potent cytotoxic molecules, we have designed and synthesized a new scaffold by
tagging quinazolinones with an acridone moiety. The new acridone-4-carboximide derivatives were
evaluated for their cytotoxic potentials against the MCF7 breast cancer cell line and three colon cancer
cell lines (LS174T, SW1398, and WiDr). Compound 26 showed relatively potent cytotoxic activity among
the derivatives, against all the cell lines tested. Mechanistic studies for the selected derivatives 7, 8, 16,
17, 25, and 26 were conducted through in vitro EGFR tyrosine kinase inhibition studies. The results
indicate that compound 26 has a better EGFR tyrosine kinase inhibitory profile. The in vitro EGFR
inhibition data was correlated with the cytotoxic properties, and molecular docking studies were
performed with regard to the receptor autophosphorylation sites of the protein kinase domain of the
EGFR.
Keywords: Acridone / Breast cancer / Docking / EGFR
Received: February 20, 2014; Revised: April 2, 2014; Accepted: April 2, 2014
DOI 10.1002/ardp.201400065
Introduction
which initiates a cascade of intracellular events [5]. Activation
of EGFR pathway triggers cell growth, differentiation, motility
and adhesion in various cell lineages [6]. In recent years, due
to the advances in the chemical and structural biology and
signaling transduction studies, EGFR was found to be a highly
promising target in oncology that is over-expressed in a
significant number of human tumors (e.g. breast, ovarian,
colon, and prostate), their expression levels often correlate
with vascularity, and is associated with poor prognosis in
patients [7–9].
Various substituted quinazoline/quinazolinone derivatives
were reported as potent inhibitors of both wild type and
mutant EGFR kinase overexpressed in various cancer cell
lines. Acridone derivatives such as GF-120918 (acridonecar-
boxamide), C1311 (imidazoacridone derivative) (Figure 1) have
very good anticancer activity in model systems [10, 11]. In
our previous studies, we have developed diverse series of
acridone derivatives with significant cytotoxic profile and also
identified an efficient pharmacophore model [12–14]. The
present investigation is focused on the design and synthesis of
Cancer is characterized by a change in controlled mecha-
nisms that manage cell proliferation and differentiation, and
is continuing to be a major health problem in developing as
well as undeveloped countries [1]. Malignancy is caused by
abnormalities in cells, which might be due to inherited genes
or caused by exposure to chemicals, radiation, or even
infectious agents [2, 3]. The epidermal growth factor receptor
(EGFR) is an important receptor tyrosine kinase involved in
many critical processes of cancer development, progression
and proliferation [4]. EGFR signaling pathway, which acts as a
major switch in different signal transduction processes,
involves active dimerization of inactive monomer EGFR
proteins followed by auto-phosphorylation of kinase domain,
Correspondence: Dr. Velivela V. S. Rajendra Prasad, Department of
Pharmaceutical Chemistry, Sitha Institute of Pharmaceutical Sciences,
Bachupally, Hyderabad, India
E-mail: drvvsrp@rediffmail.com
Fax: þ91 40 42417772
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2
Y. R. Babu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Figure 1. Structures of acridone derivatives
(GF-120918 and C1311
anticancer activity.
) showing potential
quinazolinone linked acridones as potential cytotoxic agents
against various cancer cell lines. Mechanistic studies of the
designed compounds were elucidated through in vitro
inhibition of EGFR and the result was correlated with in
silico data.
compounds produce abundant molecular ions in the form
of MþH.
Biological activity
Newly synthesized quinazolinone tagged acridone derivatives
were evaluated for their in vitro cytotoxic activity against
different cancer cell lines including the breast cancer cell line
MCF7 and the colon cancer cell lines LS174T, SW1398 and
WiDr in comparison with the reference drugs mitoxantrone,
doxorubicin and 5-fluorouracil by using sulforhodamine B
(SRB) assay. The cytotoxic data is presented in Table 3 as
IC50 concentrations expressed in micromoles (mM). Cytotoxic
results indicate that the synthesized molecules displayed
marked cytotoxic potentials against cancer cell lines. Among
the quinazolinone derivatives screened for the cytotoxic
property, bromine-containing compounds (19–27) exhibited
Results and discussion
Chemistry
Condensation of anthranilic acid with different acid chlorides
was followed by cyclization in the presence of acetic
anhydride to give 2-substituted-1,2-dihydro-4H-3,1-benzoxa-
zin-4-one derivatives (IIIa–c). Quinazoline derivatives (IVa–i,
Va–i, and VIa–i) were synthesized by the treatment of
compounds (IIIa–c) with hydrazine, thiourea, and urea,
respectively at high temperature up to 2000°C [15, 16].
Physical characterization data of quniazolinone derivatives
are listed in Table 1. Further, acridone-4-carboxylic acid (D)
and its derivatives (1–27) were synthesized according to
Scheme 1. Initially, acridone-4-carboxylic acid (D) was
synthesized by the Ullmann condensation of 2-chlorobenzoic
acid and anthranilic acid followed by cyclization with
polyphosphoric acid at 100°C [17]. The reaction of quinazo-
lines (IVa–i, Va–i and VIa–i) with acridone-4-carboxylic acid
(D) in dry toluene in the presence of pyridine, thionylchloride
and triethylamine at room temperature led to the corre-
sponding acridone-4-carboxamide derivatives (1–27). Struc-
tural features and molecular properties of acridone-4-
carboxamide derivatives are tabulated in Table 2.
All the compounds were purified by column chromatog-
raphy and dried under high vacuum up to 15 h. The purified
compounds were characterized by 1H NMR, 13C NMR and
mass spectral methods. The assignment of protons in all the
compounds is fully supported by the integration curves and
all the derivatives showed the characteristic chemical shifts
for the acridone nucleus. The mass spectra of all the
acridone derivatives were analyzed under ESI-MS. Molecular
ions were observed either in the form of Mþ and MþH in
the spectra of these acridone derivatives. From the mass
spectral data, it is clear that there is no difference in the
fragmentation pattern among the set of acridone series
compounds. In general, mass spectral features of these
compounds were similar and straight forward. Most of the
relatively better cytotoxicity. Among
a wide range of
distributed hydrophobic and higher electrostatic substitu-
tions, compounds containing phenyl at R and amide linkage
(R1) exhibited marked activity. A phenyl substituted bromo-
quinazolinone linked to acridone nucleus with an amide
linkage (compound 26) possessed higher cytotoxic potentials
than the other compounds against MCF7, LS174T and SW1398
cell lines. Further, selected compounds (7, 8, 16, 17, 25, and 26)
were tested for EGFR tyrosine kinase inhibition at
a
concentration of 10 mM. The results indicate compound 26
displayed the highest EGFR tyrosine kinase inhibition among
the selected set of ligands and the inhibition exerted by
compound 8 is also above 98%. The percentage EGFR
inhibition of the compounds was shown in Table 4. The
cytotoxic properties of these molecules showed significant
correlation with their corresponding EGFR inhibitory activity.
Molecular docking studies
In silico molecular docking studies were performed to predict
the possible mechanistic insights for these novel acridone
derivatives against EGFR. Docking of the quinazolinone
tagged acridones into the auto-phosphorylation site of
the kinase domain of EGFR (PDB ID: 1M17) revealed the
possible interacted residues. When the structural features of
the molecules were considered all the molecules consist of
adequate hydrophobic and hydrophilic regions, which are
vital during docking. Nitrogen and oxygen atoms were
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Cytotoxic Acridones as EGFR Inhibitors
3
Table 1. Physical characterization data of quinazolinone derivatives.
O
R1
R
X
N
N
Molecular
formula
Molecular
weight
Yield
(%)
Melting
point (°C)
Compound
X
R
R1
IVa
IVb
IVc
IVd
IVe
IVf
IVg
IVh
IVi
Va
Vb
Vc
Vd
Ve
Vf
Vg
Vh
Vi
VIa
VIb
VIc
VId
VIe
VIf
VIg
VIh
VIi
H
H
H
I
I
I
Br
Br
Br
H
H
H
I
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
–NH2
C9H9N3O
C14H11N3O
C15H13N3O
C9H8IN3O
C14H10IN3O
C15H12IN3O
C9H8BrN3O
C14H10BrN3O
C15H12BrN3O
C10H9N3OS
C15H11N3OS
C16H13N3OS
C10H8IN3OS
C15H10IN3OS
C16H12IN3OS
C10H8BrN3OS
C15H10BrN3OS
C16H12BrN3OS
C10H9N3O2
C15H11N3O2
C16H13N3O2
C10H8IN3O2
C15H10IN3O2
C16H12IN3O2
C10H8BrN3O2
C15H10BrN3O2
C16H12BrN3O2
175.18
237.25
251.28
301.08
363.15
377.18
254.08
316.15
330.18
219.26
281.33
295.36
345.16
407.23
421.26
298.16
360.23
374.25
203.19
265.26
279.29
329.09
391.16
405.19
282.09
344.16
358.19
58
68
70
45
78
70
40
76
70
37
42
40
40
50
53
45
40
50
33
26
50
40
35
50
45
39
40
201–203
207–209
298–300
210–213
205–209
295–296
211–213
198–200
290–292
224–226
227–229
281–283
228–230
220–223
288–290
227–229
230–233
287–289
221–223
216–218
310–312
224–226
218–220
290–294
210–212
220–223
290–292
–NH2
–CSNH2
–CSNH2
–CSNH2
–CSNH2
–CSNH2
–CSNH2
–CSNH2
–CSNH2
ꢀCSNH2
ꢀCONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
I
I
Br
Br
Br
H
H
H
I
I
I
Br
Br
Br
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
positioned appropriately in the 3D spatial arrangement of
every ligand that enhances the possibilities of better hydrogen
bonding with protein residues. In the current investigations
structural insights for protein interactions of compounds 7, 8,
16, 17, 25, and 26 are elucidated. Docking result of the dataset
compounds were listed in Table 5. Docking of compound 8, 17,
and 16 were performed into the receptor auto-phosphoryla-
tion site, which is a prominent site for most of the kinase
inhibitors. This revealed the protein ligand interaction map
this could possibly explain its higher dock score (ꢀ6.820) and
enhanced in vitro EGFR inhibition among the molecules
studied. Other residues such as Met769 and Asp831 have
shown hydrogen bond interacted with compounds 7 and 25
and oxygen atom of amide linkage in compound 25 acts
as H-bond acceptor. Larger tricyclic acridone nucleus and
quinazolinone moiety extends their contribution in docking
with their higher hydrophobic nature.
with all the possible hydrophilic and hydrophobic interac- Conclusion
tions. Compound 26 exhibited one hydrogen bond interaction
(nitrogen of quinazoline nucleus) with Lys721 (2.004 Å). It also
displayed potential hydrophobic interactions with the resi-
dues present in the binding pocket and this might be the
reason for its higher dock score (ꢀ7.015). Binding interactions
of compound 26 with EGFR is shown in Figure 2. Compound 8
did not display significant hydrogen bond interaction
(Figure 3), but it has greater hydrophobic interactions and
Two potent moieties acridone and quinazolinone individually
have shown significant cytotoxic properties. In the present
investigation we designed a new scaffold by tagging acridone
with different quinazolinones through amide bridge, with
the aim to enhance the cytotoxicity. The molecules were
evaluated for their cytotoxic activity against breast cancer
cells and three types of colon cancer cells. The results indicate
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Y. R. Babu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
COOH
COOH
Cl
i
+
N
H
H2N
COOH
COOH
C
A
B
ii
O
N
H
COOH
D
O
O
O
N
R1
R
X
SOCl2, dry pyridine
N
+
N
H
Triethylamine
N
H
O
R1
COOH
O
N
R
IV, V, VI (a–i)
D
N
X
1–27
Scheme 1. Synthesis of acridone-4-carboxamide derivatives. Reagents and conditions: (i) Cu, K2CO3, Isoamyl alcohol, reflux for 6–8 h,
160°C (ii) PPA, 100°C, 3 h.
that compound 26 showed a broad spectrum and potent
antitumor activity against MCF7 cells with IC50 of 2.9 mM. As
breast cancer cells are known to overexpress EGFR, which
leads to enhanced activation of the EGFR pathway involved in
cell proliferation, various in silico and in vitro EGFR interaction
studies further support the understanding of cytotoxic
selectivity over MCF-7 cell line and thereby help to design
new molecules. Results of the molecular docking studies
support the strong inhibitory activity of compounds 8, 17, and
26. Results of this study help in understanding the interaction
between the ligands and EGFR protein kinase in detail and
assist in the design and development of new EGFR inhibitors.
monitored by TLC and compounds were purified by using
column chromatography with silica gel Merck Grade 60 (230–
400 mesh, 60; Merck, Germany). Melting points were recorded
on a Tempirol hot-stage with microscope (AGA International,
Ambala City, Haryana, India) and are uncorrected. Elemental
analysis was performed and found values are within 0.4% of
1
theoretical values unless otherwise noted. H NMR spectra were
recorded in DMSO-d6 solution in a 5-mm tube on a Bruker drx 500
Fourier transform spectrometer (Bruker Bioscience, USA) and
TMS was used as internal standard. Chemical shifts are expressed
as d (ppm) values. The spectrometer was internally locked to
deuterium frequency of the solvent. To obtain molecular weight
information, acridone derivatives were analyzed by ESI-MS
spectrometry. Collision-induced dissociation (CID) spectra were
acquired in the positive ion mode on a MDS Sciex (Concord, Ont.,
Canada) API 4000 triple quadrupole mass spectrometry with
direct infusion of each acridone at a concentration of 10 mM in
50% methanol, at flow rate of 25 mL/min. The instrument was
operated with a spray voltage of 5.5 kV, a declustering potential of
50 eV, a source temperature of 100°C, a GSI value of 50 and the
Experimental
All chemicals and solvents were supplied by Sigma–Aldrich, India
and S.D. Fine Chemicals Limited, Mumbai. Reactions were
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Cytotoxic Acridones as EGFR Inhibitors
5
Table 2. Physical characterization data of acridone derivatives.
O
N
H
R1
O
R
O
N
N
X
Molecular
formula
Molecular
weight
Yield
(%)
Melting
point (°C)
Compound
X
R
R1
Log p
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
I
I
I
I
I
I
I
I
I
Br
Br
Br
Br
Br
Br
Br
Br
Br
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
–CH3
–C6H5
–NH
–NH
–NH
–NHCS
–NHCS
–NHCS
–NHCO
–NHCO
–NHCO
–NH
C23H16N4O3
C28H18N4O3
C29H20N4O3
C24H16N4O3S
C29H18N4O3S
C30H20N4O3S
C24H16N4O4
396.39
458.46
472.49
440.47
502.54
516.57
424.40
486.47
500.50
522.29
584.36
598.39
566.37
628.44
642.47
550.30
612.37
626.40
475.29
537.36
551.39
519.37
581.44
595.47
503.30
565.37
579.40
59
48
51
45
35
37
64
38
41
55
40
46
40
40
45
70
45
40
51
45
55
50
45
50
65
57
45
272
265
275
204
278
288
282
247
267
283
271
270
224
259
283
289
238
248
268
269
289
219
269
276
293
245
270
2.98
4.87
5.36
3.63
5.52
6.01
3.08
4.98
5.47
4.33
6.23
6.72
4.98
6.88
7.37
4.44
6.34
6.82
3.81
5.7
C29H18N4O4
C30H20N4O4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
C23H15IN4O3
C28H17IN4O3
C29H19IN4O3
C24H15IN4O3S
C29H17IN4O3S
C30H19IN4O3S
C24H15IN4O4
C29H17IN4O4
C30H19IN4O4
C23H15BrN4O3
C28H17BrN4O3
C29H19BrN4O3
C24H15BrN4O3S
C29H17BrN4O3S
C30H19BrN4O3S
C24H15BrN4O4
C29H17BrN4O4
C30H19BrN4O4
–NH
–NH
–NHCS
–NHCS
–NHCS
–NHCO
–NHCO
–NHCO
–NH
–NH
–NH
6.19
4.45
6.35
6.84
3.91
5.81
6.29
–NHCS
–NHCS
–NHCS
–NHCO
–NHCO
–NHCO
–C6H4CH3
–CH3
–C6H5
–C6H4CH3
curtain gas set at 10. Ultra-pure nitrogen was used as both curtain
gas and collision gas. MS/MS spectra of the protonated molecule
of each drug were acquired and multiple reaction monitoring
(MRM) transition for important fragments were monitored as
the collision energy was ramped from 5–100 V (step size 0.5 V).
The data for the fragment-ion curves represent an average of
five consecutive experiments.
(4mL) were stirred at room temperature in 50 mL of dry toluene for
6h. The solvent was removed under reduced pressure, and the
obtained residue was poured onto crushed ice. Product obtained
was filtered, washed with water, dried at room temperature and
recrystallized from a mixture of chloroform and ethyl acetate.
1
Yield: 81%;mp: 165–167°C; H NMR (DMSO-d6) d ppm: 2.04 (s,
3H, CH3), 7.40–8.4 (m, 4H, Ar–H), 9.23 (s, 1H, –NHCO), 11.1 (s, 1H,
OH); 13C NMR (DMSO-d6) d: 177.4, 176.3, 139.7, 138.3, 133.5, 131.9,
129.5, 128.5, 23.6; ESI-MS (m/z, %): 179.07 (100).
Chemistry
General method for the synthesis of 2-acetamidobenzoic
acid (IIa)
Quinazolinone derivatives were synthesized by using previously
described method shown in Scheme 2 [15, 16]. Anthranilic acid
(6.85g, 0.05 moles), acetyl chloride (5.1mL, 0.05 moles) and pyridine
2-Benzamidobenzoic acid (IIb)
1
Yield: 86%;mp: 270–272°C; H NMR (DMSO-d6) d ppm: 7.40–8.08
(m, 9H, Ar–H), 9.15 (s, 1H, –NHCO), 11.30 (s, 1H, OH); 13C NMR
(DMSO-d6) d: 178.3, 177.7, 141.5, 139.3, 134.7, 132.3, 129.3, 127.5,
126.4, 124.7, 123.4, 120.9; ESI-MS (m/z, %): 241.07 (95).
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Y. R. Babu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Table 3. In vitro cytotoxic activity of the compounds against
sensitive and resistant human cancer cell lines in comparison with
reference drugs doxorubicin (DX), mitoxantrone (MR), and 5-
fluorouracil (5-FU).
Table 5. Protein ligand interactions of quinazoline tagged
acridone derivatives.
No. of
Dock hydrogen
Interacted
protein
H-bond
distance (Å)
Cell linesa)/IC50 (mM) ꢁ SEMb)
Compound score
bonds
residues
7
8
16
17
25
26
ꢀ4.286
ꢀ6.820
ꢀ5.221
ꢀ6.962
ꢀ5.445
ꢀ7.015
2
–
Lys721, Met769 2.103, 1.904
Compound
MCF7
LS174T
SW1398
WiDr
–
–
–
1
2
6
7
8
9
ND
6.5 ꢁ 2.2
ND
ND
8.8 ꢁ 1.1
6.1 ꢁ 1.8
8.0 ꢁ 2.2
5.4 ꢁ 1.2
13.2 ꢁ 1.3
ND
13.8 ꢁ 3.2
ND
12.9 ꢁ 1.6
ND
1
2
1
Lys721
2.252
8.1 ꢁ 2.0
11.6 ꢁ 1.3
6.5 ꢁ 1.0
3.1 ꢁ 2.1
14.8 ꢁ 4.2
6.9 ꢁ 1.9
10.2 ꢁ 1.8
13.2 ꢁ 2.5
9.8 ꢁ 1.6
5.6 ꢁ 1.2
3.5 ꢁ 2.0
12.6 ꢁ 3.0
6.9 ꢁ 1.7
7.9 ꢁ 2.3
11.5 ꢁ 3.2
8.3 ꢁ 1.5
5.4 ꢁ 1.1
2.9 ꢁ 1.2
11.5 ꢁ 3.0
0.098
Lys721, Asp831 1.830, 2.213
Lys721 2.004
ND
13.2 ꢁ 2.9
10.7 ꢁ 1.8
9.8 ꢁ 1.4
10.5 ꢁ 2.9
14.1 ꢁ 1.8
11.2 ꢁ 2.4
ND
11.3 ꢁ 1.2
7.9 ꢁ 3.1
7.1 ꢁ 4.2
11.9 ꢁ 3.2
10.1 ꢁ 3.1
ND
10
11
12
15
16
17
18
19
20
22
23
25
26
27
DX
MR
5-FU
ND
ND
ND
11.2 ꢁ 1.0
4.4 ꢁ 1.1
11.3 ꢁ 2.0
10.6 ꢁ 2.0
10.3 ꢁ 1.8
ND
12.0 ꢁ 2.1
5.5 ꢁ 1.3
6.2 ꢁ 1.5
17.2 ꢁ 3.0
–
16.0 ꢁ 3.1
9.8 ꢁ 2.0
8.9 ꢁ 2.2
15.5 ꢁ 2.2
14.6 ꢁ 3.4
ND
18.0 ꢁ 2.1
9.2 ꢁ 1.4
6.0 ꢁ 2.0
10.5 ꢁ 2.2
–
11.4 ꢁ 1.1
8.1 ꢁ 1.9
16.9 ꢁ 2.6
18.2 ꢁ 2.2
9.4 ꢁ 2.3
ND
13.1 ꢁ 2.4
16.0 ꢁ 1.5
11.8 ꢁ 2.2
11.4 ꢁ 2.1
–
0.001
–
–
–
–
1.2 ꢁ 0.3
1.8 ꢁ 0.4
2.1 ꢁ 0.7
ND, not determined, each value represents the mean of triplicate
experiments.
a)
MCF7 breast cancer cell line, LS174T, SW1398 and WiDr are
colon cancer cells.
SEM, standard error of the mean.
b)
Figure 2. Protein ligand interactions of compound 26 with the
kinase EGFR kinase domain.
Table 4. EGFR tyrosine kinase assay of acridone derivatives at
single dose.
% Inhibition of EGFR
Compound
tyrosine kinasea)
7
8
80
98
16
86
17
91
25
90
26
100
100
98
Gefitinibb)
Staurosporinec)
a)
EGFR tyrosine kinase inhibition at concentration of 10 mM.
Percentage inhibition measured at the concentration 10 mM.
Percentage inhibition measured at the concentration 1 mM.
b)
c)
Figure 3. Hydrophobic interaction (dotted lines) of compound 8 at
the binding pocket of EGFR kinase domain.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Cytotoxic Acridones as EGFR Inhibitors
7
O
X
COOH
NH
COOH
NH2
X
X
O
AC2O
Dry toluene
RT, 6 h
ROCl
CO-R
Reflux for 3 h
N
R
Ia–c
IIIa–i
IIa–i
O
O
O
S
NH2
X
X
X
NH2NH2
NH2CSNH2
O
N
NH2
N
N
R
N
R
N
R
IIIa–i
IVa–i
Va–i
NHCHONH2
O
N
O
R
X
N
NH2
VIa–i
R: -CH3, -C6H5, -C6H4CH3
X: -H, -I, -Br
Scheme 2. Synthesis of quinazolinone derivatives.
2-(4-Methylbenzamido)benzoic acid (IIc)
2-Phenyl-4H-benzo[d][1,3]oxazin-4-one (IIIb)
Yield: 76%; mp: 291–293°C; 1H NMR (DMSO-d6) d ppm: 2.34 (s, 3H,
CH3), 7.40–8.3 (m, 8H, Ar–H), 9.73 (s, 1H, NHCO), 11.2 (s, 1H, OH);
13C NMR (DMSO-d6) d: 178.7, 177.3, 139.6, 138.4, 133.7, 131.9,
129.5, 128.3, 125.4, 124.7, 123.5, 121.2, 28.3; ESI-MS (m/z, %):
255.09 (90).
Yield: 79%; mp: 211–213°C; 1H NMR (DMSO-d6) d ppm: 7.57–8.21
(m, 9H, Ar–H); 13C NMR (DMSO-d6) d: 178.3, 141.6, 139.4, 136.2,
133.6, 132.7, 128.5, 127.3, 125.6, 124.3, 123.6, 121.3; ESI-MS
(m/z, %): 223.06 (100).
2-(p-Tolyl)-4H-benzo[d][1,3]oxazin-4-one (IIIc)
Yield: 81%; mp: 270–272°C; 1H NMR (DMSO-d6) d ppm: 2.34 (s, 3H,
CH3), 7.28–8.41 (m, 8H, Ar–H); 13C NMR (DMSO-d6) d: 179.7, 141.3,
139.5, 138.1, 134.7, 132.9, 128.5, 127.3, 125.2, 124.3, 123.7, 121.3,
28.1; ESI-MS (m/z, %): 237.08 (80).
General method for the synthesis of 2-methyl-4H-benzo[d]-
[1,3]oxazin-4-one (IIIa)
2-Acetamidobenzoic acid (1.79 g, 0.01 moles) was heated under
reflux in 10 mL of acetic anhydride under anhydrous conditions
for about 3 h. The reaction mixture was transferred into ice-cold
water and the separated solid was collected by filtration, dried
and recrystallized from ethanol.
General method for the synthesis of 3-amino-2-
methylquinazolin-4(3H)-one (IVa)
2-Methyl-4H-benzo[d][1,3]oxazin-4-one (IIIa) (1.61 g, 0.01 moles)
was heated to melt, then excess of hydrazine hydrate was added.
The mixture was fused together at 200°C in an oil bath for 1 h.
Yield: 75%; mp: 190–192°C; 1H NMR (DMSO-d6) d ppm: 2.47
(s, 3H, CH3), 7.66–8.21 (m, 4H, Ar–H); 13C NMR (DMSO-d6) d: 177.4,
142.3, 139.6, 137.3, 133.7, 131.3, 129.1, 128.7, 23.9; ESI-MS (m/z, %):
161.16 (100).
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
Y. R. Babu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
The separated solid was cooled, collected by filtration, washed
with water, dried and purified by column chromatography
(methanol/chloroform/water, 10:2:1).
141.3, 138.9, 136.3, 135.7, 133.3, 129.9, 126.9, 125.1, 123.8, 121.6,
120.3; ESI-MS (m/z, %): 265.09 (60).
Yield: 58%; mp: 201–203°C; 1H NMR (DMSO-d6) d ppm: 2.57
(s, 3H, CH3), 5.80 (s, 2H, NH2), 7.45–8.11 (m, 4H, Ar-H); 13C NMR
(DMSO-d6) d: 178.1, 142.4, 139.7, 137.3, 133.7, 131.3, 129.3, 128.7,
23.7; ESI-MS (m/z, %): 175.03 (100).
4-Oxo-2-(p-tolyl)quinazoline-3(4H)-carboxamide (VIc)
Yield: 50%; mp: 310–312°C; 1H NMR (DMSO-d6) d ppm: 3.24
(s, 3H, CH3), 5.73 (s, 2H, –NH2), 7.28–8.4 (m, 8H, Ar–H); 13C NMR
(DMSO-d6) d: 179.3, 178.1, 143.5, 138.7, 136.3, 135.1, 133.4, 129.3,
127.1, 125.9, 124.3, 123.6, 121.5, 28.1; ESI-MS (m/z, %): 279.10 (80).
3-Amino-2-phenylquinazolin-4(3H)-one (IVb)
Yield: 68%; mp: 207–209°C; 1H NMR (DMSO-d6) d ppm: 5.66
(s, 2H, –NH2), 7.49–8.20 (m, 9H, Ar–H); 13C NMR (DMSO-d6) d: 178.1,
141.3, 139.6, 136.3, 133.7, 132.5, 128.3, 127.3, 125.8, 124.7, 121.6,
120.3; ESI-MS (m/z, %): 237.09 (100).
Ullmann condensation [17]
To a mixture of o-chloro benzoic acid (0.05 moles), anthranilic
acid (0.05 moles) and copper powder (0.2 g) in 60 mL of
isoamylalcohol, 5 g of dry potassium carbonate was slowly added.
The above reaction mixture was allowed to reflux for 6–8 h on an
oil bath. After completion of the reaction as evidenced by TLC,
isoamylalcohol was removed under steam distillation. The
mixture was poured into one liter of hot water and acidified
with concentrated hydrochloric acid. The resulting precipitate
was collected by filtration, and washed with hot water. The
crude acid was dissolved in aqueous sodium hydroxide solution,
boiled in the presence of activated charcoal and filtered. On
acidification of the filtrate with concentrated hydrochloric acid,
light yellowish precipitate was obtained which was washed with
hot water and recrystallized from aqueous methanol to give
light yellow product 2,20-iminodibenzoic acid (C). Yield: 91%, mp:
187°C.
3-Amino-2-(p-tolyl)quinazolin-4(3H)-one (IVc)
Yield: 70%; mp: 298–300°C; 1H NMR (DMSO-d6) d ppm: 2.34 (s, 3H,
CH3), 4.93 (s, 2H, –NH2), 7.28–8.04 (m, 8H, Ar–H); 13C NMR
(DMSO-d6) d: 178.7, 141.3, 139.3, 137.1, 133.7, 132.4, 128.7, 127.3,
125.6, 124.3, 123.1, 121.7, 28.3; ESI-MS (m/z, %): 251.11 (90).
Synthesis of 2-methyl-4-oxoquinazoline-3(4H)-
carbothioamide (Va)
Equimolar quantities of 2-methyl-4H-benzo[d][1,3]oxazin-4-one
(IIIa) (1.61 g, 0.01 moles) and thiourea (0.76 g, 0.01 moles) were
fused together at 200°C in an oil bath for 1 h. The reaction
mixture was cooled; the precipitate was collected by filtration,
dried and crystallized from ethanol.
Yield: 37%; mp: 224–226°C; 1H NMR (DMSO-d6) d ppm: 2.34
(s, 3H, CH3), 5.56 (s, 2H, –NH2), 7.42–8.08 (m, 4H, Ar–H); 13C NMR
(DMSO-d6) d: 178.3, 176.3, 142.6, 139.1, 137.4, 133.3, 131.5, 129.3,
128.9, 23.3; ESI-MS (m/z, %): 219.05 (100).
Synthesis of acridone-4-carboxylic acid (D) [16]
Five grams of 2,20-iminodibenzoic acid (C) was taken into a round
bottom flask and 50 g of polyphosphoric acid was added and the
reaction mixture was shaken well and heated at 100°C for 3 h.
Appearance of yellow color indicated the completion of the
reaction. Then, it was poured into hot water and made alkaline by
ammonia. The yellow precipitate obtained was filtered and
collected. The acridone-4-carboxylic acid (D) was recrystallized
from acetic acid.
4-Oxo-2-phenylquinazoline-3(4H)-carbothioamide (Vb)
Yield: 42%; mp: 227–229°C; 1H NMR (DMSO-d6) d ppm: 5.84
(s, 2H, –NH2), 7.50–8.19 (m, 9H, Ar–H); 13C NMR (DMSO-d6) d: 178.7,
177.3, 141.1, 138.6, 136.7, 134.7, 132.3, 129.3, 127.9, 125.3, 124.1,
122.6, 121.3; ESI-MS (m/z, %): 281.06 (35).
Yield 69%; mp: 326–328°C; 1H NMR (DMSO-d6) d ppm: 7.29–8.48
(m, 7H, Ar–H), 11.23 (s, 1H, NH), 12.68 (s, 1H, OH); 13C NMR
(DMSO-d6) d: 178.4, 158.3, 139.5, 138.2, 133.5, 131.9, 129.5, 128.5,
125.4, 124.3, 123.4, 120.9; ESI-MS (m/z, %): 239.01 (100).
4-Oxo-2-(p-tolyl)quinazoline-3(4H)-carbothioamide (Vc)
Yield: 40%; mp: 281–283°C; 1H NMR (DMSO-d6) d ppm: 3.24 (s, 3H,
CH3), 5.86 (s, 2H, NH2), 7.31–8.24 (m, 8H, Ar–H); 13C NMR
(DMSO-d6) d: 178.7, 176.3, 141.5, 139.7, 137.3, 135.7, 133.4, 129.7,
127.5, 125.8, 124.9, 123.3, 121.9, 27.3; ESI-MS (m/z, %): 295.08 (60).
General method for the synthesis of N-(2-methyl-4-
oxoquinazolin-3(4H)-yl)-9-oxo-9,10-dihydroacridine-4-
carboxamide (1)
To the suspension of acridone (2.09 mmol) in 15 mL of dry
toluene, thionyl chloride (2.90 mmoles) and dry pyridine
(2.90 mmoles) were added. The reaction mixture was stirred at
room temperature for about 3–4 h. Then the resulting reaction
mixture was mixed with3-amino-2-methylquinazolin-4(3H)-one
(IVa) (2.09 mmoles), triethylamine (6.185 mmoles) and stirred for
another 3 h, reaction was monitored by TLC. After completion of
the reaction, the solvent was removed under vacuum, resulted
crude reaction mixture was poured into ice cold water to get
solid residue. The precipitate was filtered, washed with water and
dried. The crude product was recrystallized from methanol.
Yield 59%; mp: 172–174°C; 1H NMR (DMSO-d6) d ppm: 2.34
(s, 3H, CH3), 6.91–8.31 (m, 11H, Ar–H), 9.23 (s, 1H, CONH), 11.61
(s, 1H, NH); 13C NMR (DMSO-d6) d: 178.5, 176.2, 168.7, 145.6, 144.3,
142.4, 139.3, 138.7, 135.4, 133.5, 132.4, 129.5, 128.5, 125.4, 124.3,
123.4, 122.3, 120.9, 119.5, 23.5; ESI-MS (m/z, %): 396.12 (76); Anal.
Synthesis of 2-methyl-4-oxoquinazoline-3(4H)-
carboxamide (VIa)
Equimolar quantities of 2-methyl-4H-benzo[d][1,3]oxazin-4-one
(IIIa) (1.61 g, 0.01 moles) and urea (0.01 moles) were fused together
at 200°C in an oil bath for 1 h. The reaction mixture was cooled;
the precipitate was collected by filtration, dried and crystallized
from ethanol.
Yield: 33%; mp: 221–223°C; 1H NMR (DMSO-d6) d ppm: 2.34
(s, 3H, CH3), 5.84 (s, 2H, NH2), 7.20–8.12 (m, 4H, Ar–H); 13C NMR
(DMSO-d6) d: 178.7, 177.3, 141.6, 139.3, 137.5, 133.9, 131.3, 129.7,
128.1, 25.3; ESI-MS (m/z, %): 203.07 (60).
4-Oxo-2-phenylquinazoline-3(4H)-carboxamide (VIb)
Yield: 26%; mp: 216–218°C; 1H NMR (DMSO-d6) d ppm: 5.83 (s, 2H,
NH2), 7.52–8.34 (m, 9H, Ar–H); 13C NMR (DMSO-d6) d: 178.1, 177.4,
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
Cytotoxic Acridones as EGFR Inhibitors
9
calcd. for C23H16N4O3: C-69.69, H-4.07, N-14.13; Found: C-69.38,
H-3.78, N-13.91.
NH); 13C NMR (DMSO-d6) d: 179.7, 174.6, 168.3, 166.3, 142.8, 141.7,
139.4, 136.8, 134.2, 132.4, 130.5, 129.4, 128.4, 126.7, 124.7, 122.7,
121.3, 120.2, 119.2, 118.2, 117.8, 28.9; ESI-MS (m/z, %): 424.12 (75);
Anal. calcd. for C24H16N4O4: C-67.92, H-3.80, N-13.20; Found:
C-67.62, H-3.51, N-12.97.
N-(4-Oxo-2-phenylquinazolin-3(4H)-yl)-9-oxo-9,10-
dihydroacridine-4-carboxamide (2)
Yield 48%;mp: 165–167°C; 1H NMR (DMSO-d6) d ppm: 7.33–8.54
(m, 16H, Ar–H), 9.11 (s, 1H, NHCO), 11.6 (s, 1H, NH); 13C NMR
(DMSO-d6) d: 177.4, 176.6, 167.4, 146.4, 144.7, 143.9, 141.8, 139.8,
138.6, 137.5, 136.9, 134.6, 132.8, 129.2, 127.8, 126.7, 126.4, 124.1,
124.9, 123.4, 122.3, 120.9, 119.5, 118.6, 117.4; ESI-MS (m/z, %):
458.14 (30); Anal. calcd. for C28H18N4O3: C-73.35, H-3.96, N-12.22;
Found: C-73.04, H-3.66, N-12.01.
N-(4-Oxo-2-phenyl-3,4-dihydroquinazoline-3-carbonyl)-9-
oxo-9,10-dihydroacridine-4-carboxamide (8)
1
Yield 38%; mp: 147–149°C; H NMR (DMSO-d6) d ppm: 7.15–8.37
(m, 16H, Ar–H), 9.33 (s, 1H, NHCO), 11.91 (s, 1H, NH); 13C NMR
(DMSO-d6) d: 179.3, 174.7, 168.5, 165.3, 143.3, 142.8, 141.2, 139.6,
138.2, 136.6, 135.5, 134.3, 132.6, 130.7, 129.4, 128.6, 127.8, 126.7,
125.4, 124.7, 122.7, 121.3, 120.2, 119.2, 118.2, 117.8; ESI-MS (m/z,
%): 486.13 (65); Anal. calcd. for C29H18N4O4: C-71.60, H-3.73,
N-11.52; Found: C-71.31, H-3.43, N-11.31.
N-(4-Oxo-2-(p-tolyl)quinazolin-3(4H)-yl)-9-oxo-9,10-
dihydroacridine-4-carboxamide (3)
Yield 51%; mp: 274–277°C; 1H NMR (DMSO-d6) d ppm: 2.37 (s, 3H,
CH3), 7.33–8.55 (m, 15H, Ar–H), 9.21 (s, 1H, NHCO), 11.38 (s, 1H,
NH); 13C NMR (DMSO-d6) d: 178.4, 176.6, 168.4, 146.7, 144.5, 143.4,
142.7, 141.6, 140.4, 138.6, 136.9, 134.6, 132.8, 129.2, 127.8, 126.7,
126.4, 124.1, 124.9, 123.4, 122.3, 120.9, 119.5, 118.6, 117.4, 28.9;
ESI-MS (m/z, %): 472.15 (70); Anal. calcd. for C29H20N4O3: C-73.72,
H-4.27, N-11.86; Found: C-73.40, H-3.98, N-11.65.
N-(4-Oxo-2-(p-tolyl)-3,4-dihydroquinazoline-3-carbonyl)-9-
oxo-9,10-dihydroacridine-4-carboxamide (9)
Yield 41%; mp: 267–269°C; 1H NMR (DMSO-d6) d ppm: 2.39 (s, 3H,
CH3), 7.12–8.23 (m, 15H, Ar–H), 9.29 (s, 1H, NHCO), 11.87 (s, 1H,
NH); 13C NMR (DMSO-d6) d: 179.6, 173.3, 167.3, 165.4, 144.5, 143.8,
141.5, 139.6, 138.5, 137.3, 135.6, 134.3, 133.4, 131.5, 129.3, 128.7,
127.6, 126.3, 125.6, 123.7, 122.4, 121.7, 120.5, 119.6, 118.3, 117.6,
29.5; ESI-MS (m/z, %): 500.15 (45); Anal. calcd. for C30H20N4O4:
C-71.99, H-4.03, N-11.19; Found: C-71.67, H-3.73, N-10.97.
N-[(2-Methyl-4-oxo-3,4-dihydroquinazoline-3-
carbonothioyl)-9-oxo-9,10-dihydroacridine-4-
carboxamide (4)
N-(6-Iodo-2-methyl-4-oxoquinazolin-3(4H)-yl)-9-oxo-9,10-
1
Yield 45%; mp: 204–206°C; H NMR (DMSO-d6) d ppm: 2.35 (s, 3H,
dihydroacridine-4-carboxamide (10)
CH3), 6.91–8.37 (m, 11H, Ar–H), 9.23 (s, 1H, NHCO), 11.34 (s, 1H, NH);
13C NMR (DMSO-d6) d: 179.7, 178.2, 176.2, 164.6, 147.8, 143.1, 142.4,
141.5, 139.1, 138.4, 137.2, 135.3, 134.3, 129.4, 128.4, 127.8, 126.7,
125.4, 124.7, 122.7, 120.2. 23.5; ESI-MS (m/z, %): 440.09; Anal. calcd. for
C24H16N4O3S: C-65.44, H-3.66, N-12.72; Found: C-65.12, H-3.37, N-12.51.
1
Yield 55%; mp: 180–182°C; H NMR (DMSO-d6) d ppm: 2.34 (s, 3H,
CH3), 6.91–8.31 (m, 10H, Ar–H), 9.23 (s, 1H, CONH), 11.41 (s, 1H, NH);
13C NMR (DMSO-d6) d: 178.3, 176.9, 168.1, 145.7, 144.3, 141.4, 139.3,
138.7, 136.4, 133.1, 132.6, 129.7, 128.3, 125.4, 124.3, 123.6, 122.7,
121.9, 119.3, 23.3; ESI-MS (m/z, %): 523.29 (76); Anal. calcd. for
C23H15IN4O3: C-52.89, H-2.89, N-10.73; Found: C-52.57, H-2.57, N-10.51.
N-(4-Oxo-2-phenyl-3,4dihydroquinazoline-3-
carbonothioyl)-9-oxo-9,10-dihydroacridine-4-
carboxamide (5)
N-[(6-Iodo-2-methyl-4-oxoquinazolin-3(4H)-yl)carbonyl]-9-
1
oxo-9,10-dihydroacridine-4-carboxamide (16)
Yield 35%; mp: 178–181°C; H NMR (DMSO-d6) d ppm: 7.18–8.38
Yield 70%; mp: 180–182°C; 1H NMR (DMSO-d6) d ppm: 2.32 (s, 3H,
CH3), 6.91–8.37 (m, 10H, Ar–H), 10.23 (s, 1H, NHCO), 11.56 (s, 1H,
NH); 13C NMR (DMSO-d6) d: 179.3, 177.6, 168.7, 166.9, 142.3, 141.7,
139.1, 136.3, 135.2, 132.4, 130.3, 129.4, 128.3, 126.7, 124.1, 122.7,
121.3, 120.2, 119.3, 118.2, 117.8, 28.7; ESI-MS (m/z, %): 551.3 (75);
Anal. calcd. for C24H15IN4O4: C-52.38, H-2.75, N-10.18; Found:
C-52.37, H-2.43, N-9.97.
(m, 16H, Ar-H), 9.13 (s, 1H, NHCO), 11.23 (s, 1H, NH); 13C NMR
(DMSO-d6) d: 179.1, 173.3, 168.3, 167.3, 143.3, 142.8, 141.2, 139.6,
138.2, 136.6, 135.2, 134.2, 132.4, 130.5, 129.6, 128.6, 127.2, 126.5,
125.8, 124.7, 123.3, 121.3, 120.2, 119.4, 118.6, 117.8; ESI-MS
(m/z, %): 502.11 (65); Anal. calcd. for C29H18N4O3S: C-69.31, H-3.61,
N-11.15; Found: C-69.02, H-3.07, N-10.94.
N-(4-Oxo-2-(p-tolyl)-3,4-dihydroquinazoline-3-
carbonothioyl)-9-oxo-9,10-dihydroacridine-4-
carboxamide (6)
N-(6-Bromo-2-methyl-4-oxoquinazolin-3(4H)-yl)-9-oxo-
9,10-dihydroacridine-4-carboxamide (19)
Yield 51%; mp: 170–172°C; 1H NMR (DMSO-d6) d ppm: 2.34 (s, 3H, CH3),
6.91–8.31 (m, 10H, Ar–H), 9.23 (s, 1H, CONH), 11.51 (s, 1H, NH);
13C NMR (DMSO-d6) d: 178.2, 176.1, 168.3, 145.9, 144.4, 142.1, 139.3,
137.7, 136.4, 133.1, 132.3, 129.5, 128.3, 125.1, 124.3, 123.7, 122.3, 120.3,
119.6, 23.7; ESI-MS (m/z, %): 476.2 (76); Anal. calcd. for C23H15BrN4O3:
C-58.12, H-3.18, N-11.70; Found: C-57.80, H-2.86, N-11.47.
1
Yield 37%; mp: 288–290°C; H NMR (DMSO-d6) d ppm: 2.39 (s, 3H,
CH3), 6.85–8.33 (m, 15H, Ar–H), 9.23 (s, 1H, NHCO), 11.63 (s, 1H, NH);
13C NMR (DMSO-d6) d: 179.2, 176.1, 171.3, 168.3, 146.7, 145.2, 143.3,
142.8, 141.2, 139.6, 138.2, 136.6, 135.2, 134.2, 132.4, 130.5, 129.4,
128.4, 127.8, 126.7, 125.4, 124.7, 122.7, 120.2, 119.2, 118.2, 117.8,
25.1; ESI-MS (m/z, %): 516.13 (80); Anal. calcd. for C30H20N4O3S: C-
69.75, H-3.90, N-10.85; Found: C-69.44, H-3.59, N-10.62.
N-[(6-Bromo-2-methyl-4-oxoquinazolin-3(4H)-yl)-
N-[(2-Methyl-4-oxo-3,4-dihydroquinazoline-3-carbonyl)-9-
carbonyl]-9-oxo-9,10-dihydroacridine-4-carboxamide (25)
Yield 65%; mp: 188–190°C; 1H NMR (DMSO-d6) d ppm: 2.32 (s, 3H,
CH3), 6.91–8.37 (m, 10H, Ar–H), 10.23 (s, 1H, NHCO), 11.56 (s, 1H,
NH); 13C NMR (DMSO-d6) d: 179.5, 176.6, 168.7, 166.9, 142.3, 141.7,
oxo-9,10-dihydroacridine-4-carboxamide (7)
Yield 64%; mp: 182–185°C; 1H NMR (DMSO-d6) d ppm: 2.32 (s, 3H,
CH3), 6.91–8.37 (m, 11H, Ar–H), 10.23 (s, 1H, NHCO), 11.56 (s, 1H,
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Y. R. Babu et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–11
139.7, 136.3, 134.9, 132.6, 130.5, 129.4, 128.4, 126.3, 124.9, 122.6,
121.5, 120.3, 119.7, 118.2, 117.3, 28.3; ESI-MS (m/z, %): 504.3 (75);
Anal. calcd. for C24H15BrN4O4: C-57.27, H-3.00, N-11.13; Found:
C-56.96, H-2.71, N-10.92.
Molecular docking studies
Initially a digital structure of the target kinase domain of EGFR
was retrieved from the Protein databank website with PDB Id:
1M17. Protein structure optimization was done by using Protein
preparation wizard, which corrects the protein structure by
adding hydrogen’s to satisfy the valences and optimized by
using OPLS-2005 force field (optimized potentials for liquid
simulations) [20].
Pharmacology
In vitro cytotoxic studies by SRB assay
The acridone tagged quinazolinone derivatives were evaluated
for cytotoxicity against different cancer cells (MCF7, LS174T,
SW1398, and WiDr) by using the sulforhodamine B (SRB) [18]. In
brief, cells were cultured in RPMI 1640 supplemented with 10%
fetal calf serum, and cultures were passed once or twice a week
using trypsin EDTA to detach the cells from their culture flasks.
The fast growing cells were harvested, counted, and plated at
suitable concentrations in 96-well micro plates. After incubation
for 24 h, the compounds dissolved in the culture medium were
added to the culture wells in triplicate and incubated further for
72 h at 37°C under 5% CO2 atmosphere. One separate plate was
used to determine the number of cells at the moment of drug
addition. This plate was immediately fixed. The other cultures
were fixed at 72 h with cold TCA and stained with 0.4% SRB
dissolved in 1% acetic acid. After dissolving the bound stain with
150 ml of 10 mM unbuffered Tris base (Tris (hydroxyl methyl)
amino methane) solution using gyratory shaker, absorbance
was measured at 540 nm using a microplate reader (Tecan).
The cytotoxicity was assessed by measuring the concentration
required to inhibit protein synthesis by 50% (i.e. IC50) as
comparison. Each value represents the mean of triplicate
experiments.
Protein ligand interactions were examined by using Glide XP
docking protocol, which includes two basic steps, Receptor
Grid generation and ligand docking. Previous X-ray crystallo-
graphic studies of the EGFR complex revealed the binding site
components and a grid was generated considering the volume of
the active binding pocket of the protein. Each ligand was docked
in to the active site of the target protein using XP mode of Glide.
Glide Score includes a steric-clash term, adds polar terms featured
by Schrodinger to correct electrostatic mismatches [21–24].
Glide score is a combination of hydrophilic, hydrophobic, metal
binding groups, Van der Waals energy, freezing rotatable bonds,
and polar interactions with receptor.
V.V.S.R.P. would like to gratefully acknowledge All India Council for
Technical Education (AICTE), Government of India for “Career Award
for Young Teachers” (CAYT) and research grant (1-51/RIFD/CA/11/2011-
12). The authors would like to acknowledge the Laboratory Medical
Oncology, VU University Medical Center, Amsterdam, The Netherlands
for providing necessary research facilities to carry out the cytotoxicity
studies.
The authors have declared no conflict of interest.
In vitro inhibition of EGFR tyrosine kinase
The EGFR tyrosine kinase inhibition assay was performed by
using Kinase-Glo Plus luminescence kinase assay kit. In this
method, kinase activity was determined by quantitating the
amount of ATP remaining in solution of a kinase reaction [19]. The
luminescent signal from the assay is correlated with the amount
of ATP present and it was inversely related with kinase activity.
The compounds were diluted to 100 mM in 10% DMSO and 5 mL
of the dilution was added to a 50 mL reaction. All of the enzymatic
reactions were conducted at 30°C for 40 min, 50 mL of reaction
mixture contains 10 mM MgCl2, 40 mM Tris, pH 7.4, 0.1 mg/mL
BSA, 0.2 mg/mL Poly (Glu, Tyr) substrate, 10 mM ATP and EGFR.
After the enzymatic reaction, 50 mL of Kinase-GloPlus Lumines-
cence kinase assay solution was added to each reaction and
incubate the plate for 5 min at room temperature. The protein
kinase assays used to determine IC50 value were performed using
ADP-Glo assay kit, which measures the generation of ADP by the
protein kinase. Generation of ADP by the protein kinase reaction
leads to an increase in luminescence signal in the presence of
ADP-Glo assay kit. The assay was started by incubating the
reaction mixture in a 96-well plate at 30°C for 30 min. After
the 30 min incubation period, the assay was terminated by the
addition of 25 mL of ADP-Glo reagent. The 96-well plate was
shaken and then incubated for 30 min at ambient temperature.
Then, 50 mL of kinase detection reagent was added, the 96-well
reaction plate was then read using the ADP-Glo Luminescence
reader. Blank control was set up that included all the assay
components except the addition of appropriate substrate. The
corrected activity for each protein kinase target was determined
by removing the blank control value.
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