2-(Chloromethyl)-3-phenylquinazolin-4(3H)-ones as potent anticancer agents; cytotoxicity, molecular docking and in silico studies

Article abstract of DOI:10.1007/s13738-021-02168-1

Abstract: In order to show antiproliferation and cancerous cell growth inhibition of quinazoline derivatives, a series of 2-(chloromethyl)-3-phenylquinazolin-4(3H)-ones (H₁–H₁₁) were synthesized. In vitro cytotoxic activities were evaluated against three human cancer cell lines: A549, MCF-7 and SW1116 using colorimetric MTT assay. Comparing their effects together and with cisplatin as a positive control indicated that H₃, H₅ and H₆ exhibited better antitumor activities on A549 cell line with IC₅₀ values less than 10?μM versus 12?μM for cisplatin. In the case of MCF-7 and SW1116 cell lines, almost all compounds displayed better cytotoxic activities than cisplatin. Molecular docking studies were applied on epidermal growth factor receptor (EGFR) as the main target of quinazoline scaffolds in cancer therapy to predict the binding energies, binding modes and orientation of these ligands toward the active site of the receptor. In silico physicochemical parameters and ADMET profiling calculations also were done. All compounds showed lower binding energies than erlotinib, the inhibitor of EGFR. Taken together, our findings showed potential anticancer effect of quinazoline compounds bearing various phenyl ring substitutions. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s13738-021-02168-1

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-021-02168-1
ORIGINAL PAPER
2‑(Chloromethyl)‑3‑phenylquinazolin‑4(3H)‑ones as potent anticancer
agents; cytotoxicity, molecular docking and in silico studies
Leila Emami1,2 · Zeinab Faghih1 · Soghra Khabnadideh1,2 · Zahra Rezaei1,2 · Razieh Sabet2 · Ebrahim Harigh1,2
Zahra Faghih3
·
Received: 18 October 2020 / Accepted: 2 January 2021
© Iranian Chemical Society 2021
Abstract
In order to show antiproliferation and cancerous cell growth inhibition of quinazoline derivatives, a series of 2-(chloromethyl)-
3-phenylquinazolin-4(3H)-ones (H1–H11) were synthesized. In vitro cytotoxic activities were evaluated against three human
cancer cell lines: A549, MCF-7 and SW1116 using colorimetric MTT assay. Comparing their efects together and with
cisplatin as a positive control indicated that H3, H5 and H6 exhibited better antitumor activities on A549 cell line with IC50
values less than 10 μM versus 12 μM for cisplatin. In the case of MCF-7 and SW1116 cell lines, almost all compounds dis-
played better cytotoxic activities than cisplatin. Molecular docking studies were applied on epidermal growth factor receptor
(EGFR) as the main target of quinazoline scafolds in cancer therapy to predict the binding energies, binding modes and
orientation of these ligands toward the active site of the receptor. In silico physicochemical parameters and ADMET profl-
ing calculations also were done. All compounds showed lower binding energies than erlotinib, the inhibitor of EGFR. Taken
together, our fndings showed potential anticancer efect of quinazoline compounds bearing various phenyl ring substitutions.
* Zeinab Faghih
faghihl@sums.ac.ir; layafaghih@gmail.com
* Zahra Faghih
faghihz@sums.ac.ir
1
Pharmaceutical Sciences Research Center, Shiraz University
of Medical Sciences, Shiraz, Iran
2
Department of Medicinal Chemistry, School of Pharmacy,
Shiraz University of Medical Sciences, Shiraz, Iran
3
Shiraz Institute for Cancer Research, Medical School, Shiraz
University of Medical Sciences, Shiraz, Iran
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
Graphic abstract
Keywords Quinazoline · Cytotoxicity · EGFR · Molecular docking
metastasis and survival [10]. EGFR is overexpressed in
numerous tumors, such as lung, brain, ovarian, colon,
breast, head and prostate tumors [11, 12] Erlotinib and
geftinib are the most potent and selective EGFR inhibi-
tors which have quinazoline scafold in their structures.
Besides, various quinazolinone analogs (I–III) have been
proved to be efective as anticancer agents [2, 13–15].
Recently, there are some reports about synthesis and anti-
cancer activities of quinazoline derivatives. For example, in
2018, Sherihan El-Sayed et al. synthesized quinazolinone-
based rhodanine derivatives (IV) which displayed appropri-
ate IC50 values in the range of 10–60 µM. Their SAR studies
revealed that bulky, hydrophobic and electron-withdrawing
substituents at para-position of the N3-phenyl ring increased
the anticancer activity [16]. Fadhil Lafta Faraj et al. synthe-
sized novel quinazolinone-Schif bases (V) which possessed
remarkable cytotoxic activity against breast cancer cell line,
MCF-7 [17]. A new series of 2-aryl-4-(aryl amino) quina-
zoline (VI) were synthesized and screened for anticancer
activity by Rahmannejadi et al. in 2018 [18]. Some of these
compounds indicated desirable IC50 values toward lung
Introduction
Cancer is a generic term for a variety of diseases that
causes cells to divide uncontrollably which damage the
afected organ. There are many problems in cancer treat-
ment mainly because of diversity of the tumor types and
the similarities between tumor and normal cells. Thus, dis-
covering and designing new anticancer agents with specifc
afnity to tumor cells seems to be necessary. Quinazoline
scafolds and their bioisosteres are well known as a useful
and important scafold in drug discovery. These privileged
structures attract medicinal chemists through their broad
spectrum of biological activities such as antidiabetic [1],
anticancer [2], antihistaminic [3], anti-infammatory [4],
antibacterial [5], antifungal [6] and antiviral activities [7].
Taking a deep look at the masses of cancer-causing
mechanisms, it can be admitted the epidermal growth
factor receptor (EGFR) plays an important role in cell
growth regulation. It is also considered as one of the most
attractive targets of tyrosine kinase (TK) inhibitors [8, 9]
which play vital roles in cell proliferation, diferentiation,
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Journal of the Iranian Chemical Society
adenocarcinoma (A549) and ovarian cancer (SKOV3) cell
line. Also, recently, a series of bis‐quinazoline‐4(3H)‐one’s
derivatives (VII) were reported, which represented desired
inhibitory activities against three human tumor cell lines,
including A549, MCF‐7 and SKOV3 cell lines, and the SAR
studies showed that analogues with bearing mono-bromo
group at R1 position indicated remarkable cytotoxic activity,
especially against A549 and MCF‐7 cell lines. (Fig. 1) [19].
Regarding the current progressive interest on develop-
ing anticancer agents based on quinazoline scafold, cyto-
toxity activity and continuing our recent projects on them
(VIII, IX) [20, 21], here we prepared some quinazolinone
derivatives (Fig. 2), to have several 2-chloromethyl-3-sub-
stituted phenylquinazolin-4(3H)-ones containing diferent
electron-poor and electron-donating groups on phenyl ring
at positions 3 (N-3) and also C-2 chloro-methylene group
on quinazoline moiety. We then investigated their potential
antitumor activities and the efect of diferent substitutions
on their activities through structure–activity relationship
(SAR) analysis. In addition, molecular docking simulation
was performed to explore the binding energies, modes and
the most appropriate orientation of the synthesized com-
pounds as ligands toward EGFR, compared with those for
erlotinib, a known inhibitor and internal ligand of EGFR. In
silico physicochemical parameters (ADME) prediction of all
compounds and cisplatin was done on the basis of Lipinski
and Veber rules.
Materials and methods
Chemistry
All solvents and chemicals were obtained from Merck com-
pany (Germany) in analytical and/or synthetic grade and
used without further purifcation. All yields refer to isolated
products after purifcation method. 2-Chloromethyl-3-substi-
tuted phenylquinazolin-4(3H)-one derivatives were prepared
and synthesized (H1–H11) according to the method described
in the literature [22–25]. Melting points were determined
using Electrothermal 9200 apparatus (Electrothermal,
UK). Infrared spectra were run using potassium bromide
(KBr) pressed pellet method on a VERTEX70 spectrom-
eter (Bruker, Germany). 1HNMR and 13CNMR spectra were
recorded on a Bruker DRX-400 Avance instrument using
CDCl3 as deuterated solvent, with an internal standard of
tetramethylsilane at 400 and 100 MHz, respectively. Mass
spectra were obtained on Mass instrument using (M++1)
mode (Agilent Technologies, USA). Spots on developed
thin-layer chromatography (TLC) were detected under UV
light.
Fig. 1 Some recently reported
O
R2
quinazoline-based anticancer
agents
R1
HO
N
O
O
O
N
NH
N
N
R1
S
R1
S
R2
N
H
HO
IV
V
R2
X
O
N
O
Cl
HN
NH
HN
2R2
R
R1
N
R1
N
H
N
R
VII
VI
O
O
Br
NH
NH
N
N
R
R
Br
IX
VIII
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Journal of the Iranian Chemical Society
Fig. 2 Designed target com-
pounds based on marketed
drugs and some reported anti-
cancer agents
O
O
O
DIPEA, r.t
O
OH
NH2
Cl
+
Cl
Cl
CH3CN, 2h
General procedure for synthesis
N
C
of 2‑(chloromethyl)‑4H‑benzo[d][1,3] oxazin‑4‑one
(C)
A
B
Scheme 1 Schematic of 2-(chloromethyl)-4H-benzo[d][1,3] oxazin-
To provide a solution of 2-(chloromethyl)-4H-benzo[d][1,3]
oxazin-4-one, 1 mmol of anthranilic acid (A) in (10 ml) dichlo-
romethane and 1.5 mmol of diisopropylethylamine (DIPEA)
were added. Then, 1.2 mmol of chloroacetyl chloride (B) was
added dropwise for 20 min and the resulting mixture was
stirred at room temperature for 2 h. After that, the mixture
was extracted using ethyl acetate (2×20 ml), and the organic
layer was washed with water and dried over anhydrous sodium
sulfate (Na2SO4), fltered and concentrated under vacuum to
aford the 2-(chloromethyl)-4H-benzo[d][1,3] oxazin-4-one
(Scheme 1).
4-one (C) synthesis
General procedure for synthesis
of 2‑chloromethyl‑3‑substituted
phenylquinazolin‑4(3H)‑one derivatives (H1–H11)
The intermediate compound (C) was treated with 1 mmol of
diferent substituted anilines (D1–D11) and 1.5 mmol of PCl3
in acetonitrile (CH3CN) at 60 °C for 2 h. When the reac-
tion was completed, the mixture was basifed with saturated
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Journal of the Iranian Chemical Society
(d, 2H, J = 0.8 CH2Cl). 13C-NMR (100 MHz, CDCl3) δC
(ppm)=161.44, 150.25, 146.34, 135.9, 134.6, 132.0, 129.9,
127.8, 127.4, 126.74, 126.3, 126.3, 125.6, 125.6, 120.6,
43.1.
O
N
O
R
R
60 oC
PCl ,
3
N
O
+
Cl
Cl
CH3CN, 2h
H2N
N
C
H1-H11
D1-D11
R1:3-Cl
R2:4-Cl
R3:4-F
R5:4-Phenoxy R9:3,4-diMe
R6:3-F, 4-Me R10:5-Cl, 2-OMe
R7:3-F, 4-Me R11:2,4-diOMe
2‑(chloromethyl)‑3‑(4‑phenoxyphenyl)
quinazolin‑4(3H)‑one (H5)
R4:3-CF3 R8:5-Cl, 2-Me
Cream powder; yield 73%; m.p. 175 °C; IR (KBr) v
(cm−1): 3097–3040 (C–H, aromatic), 2975(C–H, aliphatic),
1688 (C=O, stretch), 1597 (C=C aromatic), 1501 (C=N,
stretch), 1H-NMR (400 MHz, CDCl3) δH (ppm)=8.32 (dd,
1H, J = 7.2 Hz, 0.8 Hz, aromatic), 7.78–7.86 (m,2H, aro-
matic), 7.56 (t, 1H, J=8 Hz, aromatic), 7.43 (t, 2H, J=8 Hz,
aromatic), 7.32 (d, 2H, J= 8.8 Hz, aromatic), 7.22 (t, 1H,
J=7.2 Hz, aromatic), 7.15–7.17 (m, 4H, aromatic), 4.34(s,
2H, CH2Cl). 13C-NMR (100 MHz, CDCl3) δC (ppm)=162.3,
158.8, 155.8, 151.7, 146.9, 134.8, 130.2, 130.0, 127.9,
127.8, 127.2, 124.4, 121.3, 120.0, 118.87, 43.7. MS m/z
(%):362 (650), 313 (100), 234 (27), 77 (55), 114.9 (26).
Scheme 2 Schematic of 3-substituted quinazolin-4(3H)-one deriva-
tives (H1–H11) synthesis
NaHCO3 solution and extracted by ethyl acetate (3×20 ml),
dried over anhydrous Na2SO4 and fltered. After removing
solvent, a purifed product was obtained through recrystal-
lization of the crude product in hot ethanol (Scheme 2). All
the chemical structures were elucidated by diferent spec-
troscopic methods such as Mass and NMR except for H1,
H2, H3 and H9 which were confrmed by comparing their
melting points with previously reported data (Table 2).
3‑(3‑chloro‑4‑fuorophenyl)‑2‑(chloromethyl)
quinazolin‑4(3H)‑one (H6)
Spectra data for ligand
White powder; yield 75%; m.p. 187 °C; IR (KBr) v (cm−1):
3046–3020 (C–H, aromatic), 2960–2858(C–H, aliphatic),
1658 (C=O, stretch), 1587 (C=C aromatic), 1498 (C=N,
2‑(chloromethyl)‑3‑(3‑(trifuoromethyl) phenyl)
quinazolin‑4(3H)‑one (H1)
1
stretch). H-NMR (400 MHz, CDCl3) δH (ppm) = 8.29
(dd, 1H, J=8 Hz, 1.2 Hz, aromatic), 7.85 (t, 1H, J=8 Hz,
aromatic), 7.78 (d, 1H, J= 8 Hz, aromatic), 7.58 (m, 1H,
J=8 Hz, aromatic), 7.49 (dd, 1H, J=8.8 Hz, 2.4 Hz, aro-
matic), 7.32–7.39 (m, 2H, aromatic), 4.39 (s, 2H, CH2Cl).
13C-NMR (100 MHz, CDCl3) δC (ppm) = 161.9, 160.0,
157.5, 150.8, 146.8, 135.1, 131.4, 129.0, 129.0, 128.3,
127.8, 127.2, 117.7, 117.5, 43.5. MS m/z (%):0.317(7), 302
(302). 253 (68), 140 (100), 134 (65), 119.9 (50), 77 (30).
Cream powder; yield 87%; m.p. 115 °C.
2‑(chloromethyl)‑3‑(4‑chlorophenyl)
quinazolin‑4(3H)‑one (H2)
White crystal; yield 86%; m.p. 173 °C.
2‑(chloromethyl)‑3‑(4‑fuorophenyl)
quinazolin‑4(3H)‑one (H3)
2‑(chloromethyl)‑3‑(3‑fuoro‑4‑methylphenyl)
quinazolin‑4(3H)‑one (H7)
White-yellow solid; yield 83%; m.p. 176 °C.
White powder; yield 64%; m.p. 149 °C; IR (KBr) v (cm−1):
3062–3026 (C–H, aromatic), 2995–2925(C–H, aliphatic)
1697 (C=O, stretch), 1602 (C=C aromatic), 1504 (C=N,
stretch). 1H-NMR (400 MHz, CDCl3) δH (ppm)=8.31 (d,
1H, J=8 Hz, aromatic), 7.78–7.86 (m, 2H, aromatic), 7.57
(t, 1H, J=8 Hz, aromatic), 7.41 (t, 1H, J=8 Hz aromatic),
7.09 (d, 2H, J=8.8 Hz aromatic), 4.31 (s, 2H, CH2Cl), 2.4 (s,
3H, CH3). 13C-NMR (100 MHz, CDCl3) δC (ppm)=162.5,
161.9, 160, 151.3, 146.9, 134.9, 132.4, 128, 127.8, 127.2,
124.2, 121.2, 116.1, 115.8, 43.5, 14.6. MS m/z (%): 302
(47), 253 (100), 267 (25), 108.9 (23).
2‑(chloromethyl)‑3‑(3‑(trifuoromethyl) phenyl)
quinazolin‑4(3H)‑one (H4)
White crystal; yield 91%; m.p. 153 °C; IR (KBr) v (cm−1):
3069–3021 (C–H, aromatic), 2971 (C–H, aliphatic), 1686
(C=O, stretch), 1605 (C=C aromatic), 1572 (C=N, stretch),
1H-NMR (400 MHz, CDCl3) δH (ppm) = 8.30–8.32 (m,
1H, aromatic), 7.86 (s, 1H, aromatic), 7.84 (d, J=1.6 Hz,
1H, aromatic), 7.80–7.82 (m, 1H, aromatic), 7.76 (t, 1H,
J = 8.8 Hz, aromatic), 7.69 (s, 1H, aromatic), 7.61–7.64
(m, 1H, aromatic), 7.57–7.58 (m, 1H, aromatic), 4.27
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Journal of the Iranian Chemical Society
99.7, 55.8, 55.6, 43.8. MS m/z (%): 330 (33), 299 (45), 281
(100), 76 (15).
3‑(5‑chloro‑2‑methylphenyl)‑2‑(chloromethyl)
quinazolin‑4(3H)‑one (H8)
White powder; yield 79%; m.p. 157 °C; IR (KBr) v (cm−1):
3058–3025 (C–H, aromatic), 2986 (C–H, aliphatic), 1685
(C=O, stretch), 1598 (C=C aromatic), 1509 (C=N, stretch).
1H-NMR (400 MHz, CDCl3) δH (ppm) = 8.33 (d, 1H,
J=8 Hz, aromatic), 7.80–7.88 (m, 2H, aromatic), 7.58 (t,
1H, J=6.8 Hz, aromatic), 7.44–7.47 (m, 1H, aromatic), 7.38
(d, 1H, J=8.4 Hz, aromatic), 7.29–7.31 (m, 1H, aromatic),
4.29 (d, 1H, J = 15 Hz, CH2Cl), 4.19 (d, 1H, J = 15 Hz,
CH2Cl), 2.15(s, 3H, CH3). 13C-NMR (100 MHz, CDCl3) δC
(ppm)=161.1, 150.9, 147.0, 135.9, 135.1, 135.1, 132.5, 130.3,
128.8, 128.1, 127.9, 127.3, 125.5, 121.0, 43.5, 17.4.
Cytotoxicity assays
Cell line and cell culture
Three human cancer cell lines, A549 (non-small cell lung
carcinoma), MCF-7 (breast carcinoma) and SW1116
(colon carcinoma), were obtained from National Cell Bank
of Iran (NCBI, Pasteur Institute, Tehran, Iran). All cell
lines were cultured in complete culture media containing
RPMI 1640, 10% fetal bovine serum (FBS) and 1% peni-
cillin–streptomycin (all from Gibco, USA) at 37 °C in a
humidifed CO2 incubator.
2‑(chloromethyl)‑3‑(3,4‑dimethylphenyl)
quinazolin‑4(3H)‑one (H9)
Yellow powder; yield 69%; m.p. 127 °C.
MTT assay
Cytotoxic activities of 11 derivatives of quinazolin-
4-(3H)-one were determined by a standard 3-(4,5dimethyl-
thiazol-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay
as reported previously [26, 27]. The cells were seeded in
96-well microplates at a density of 1×104 cells per well in
100 μl of complete culture medium for MCF-7 and 8×103
cells per well for A549 and SW1116 cell lines. After 24 h,
each cell was treated with six diferent concentrations of
the compounds ranging from 1 to 200 μM in triplicate.
The fnal dimethyl sulfoxide (DMSO) (Sigma-Aldrich,
Germany) concentration, as the solvent, tried to be kept
less than 0.1%. Diferent concentrations of cisplatin were
also used as positive control (1–100 µM). Three untreated
wells were considered as negative controls. After 72 h, the
media were replaced with 100 μl fresh media containing
0.5 mg/l of MTT solution. The plate was then returned
to the incubator for more 4 h until intracellular purple
formazan crystals were appeared. The media were then
discarded, and 150 μl DMSO was added and incubated at
37 °C in the dark for 10 min to dissolve the crystals. The
absorbance of individual well was then read at 490 nm by
ELISA reader. To estimate and analyze the data, Excel
2013 and Curve Expert 1.4. were applied. The following
formula was used to measure the percentage of inhibitory
efect for each compound:
3‑(5‑chloro‑2‑methoxy phenyl)‑2‑(chloromethyl)
quinazolin‑4(3H)‑one (H10)
Cream powder; yield 83%; m.p. 162 °C; IR (KBr) v (cm−1):
3091–3001 (C–H, aromatic), 2979–2842 (C–H, aliphatic),
1690 (C=O, stretch), 1603 (C=C aromatic), 1498 (C=N,
1
stretch). H-NMR (400 MHz, CDCl3) δH (ppm)=7.31 (d,
1H, J=8 Hz, aromatic), 7.78–7.86 (m, 2H, aromatic), 7.56
(t, 1H, J=8 Hz, aromatic), 7.50 (dd, 1H, J=8.8 Hz, 2.4 Hz,
aromatic), 7.38 (d, 1H, J=2.4 Hz aromatic), 7.05 (d, 1H,
J=8.8 Hz, aromatic), 4.38 (d, 1H, J=12 Hz, CH2Cl), 4.19 (d,
1H, J=12 Hz, CH2Cl), 3.8 (s, 3H, CH3). 3.8 (s, 3H, OCH3).
13C-NMR (100 MHz, CDCl3) δC (ppm)=161.5, 153.5, 151.6,
147.0, 134.9, 131.3, 130.5, 127.9, 127.7, 127.3, 125.8, 125.3,
121.2, 113.2, 56.2, 43.6. MS m/z (%): 334 (23), 303 (92), 285
(100), 76 (30).
2‑(chloromethyl)‑3‑(2,4‑dimethoxyphenyl)
quinazolin‑4(3H)‑one (H11)
Gray powder; yield 76%; m.p. 174 °C; IR (KBr) v (cm−1):
3106–3023 (C–H, aromatic), 2990–2844 (C–H, aliphatic)
1693 (C=O, stretch), 1614 (C=C aromatic), 1508 (C=N,
1
stretch). H-NMR (400 MHz, CDCl3) δH (ppm)=8.32 (d,
[(
) (
)
]
1H, J=7.6 Hz, aromatic), 7.78–7.84 (m, 2H, aromatic), 7.54
(t, 1H, J=8 Hz, aromatic), 7.27 (d, 1H, J=8.8 Hz aromatic),
6.64–6.68 (m, 2H, aromatic), 4.37 (d, 1H, J=12 Hz, CH2Cl),
4.19 (d, 1H, J=12 Hz, CH2Cl), 3.9 (s, 3H, OCH3), 3.77 (s,3H,
OCH3)13C-NMR (100 MHz, CDCl3) δC (ppm)=162.0, 155.5,
152.7, 147.2, 134.6, 130.8, 127.6, 127.3, 121.4, 117.3, 104.9,
IC = 100− ODtest−ODblank ∕ ODnegative × 100
A plot of the IC versus concentration was delineated and
IC50, demonstrating the 50% growth inhibition of the cells
was obtained. The data were expressed as the mean SD
for each analysis. Statistical analyses were performed by
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Journal of the Iranian Chemical Society
Table 1 In vitro cytotoxic activity of 2-(chloromethyl)-3-phe-
procedure. Then, binding interactions between docked
compounds and receptor were analyzed using AutoDock
tools program (ADT; version 1.5.6) and PLIP (fully auto-
mated protein–ligand interaction profler) [32].
nylquinazolin-4(3H)-one derivatives
Entry
IC50 SD (µM)*
A-549
MCF-7
SW1116
4.5 0.3
H1
17.0 1.9
14.8 1.7
5.7 0.6
72.5 5.0
10.0 0.4
9.3 1.1
13.2 0.2
14.0 1.4
13.0 0.5
23.0 1.8
22.5 3.5
12.0 1.9
10.6 1.6
14.9 1.5
23.4 1.6
131.1 12.1
5.4 0.6
5.8 0.4
7.8 1.2
9.7 0.7
11.5 1.6
9.4 1.4
24.4 1.6
14.9 2.1
In silico physicochemical parameters and ADMET
profling calculations
H2
8.2 0.4
6.1 0.2
112.0 3.1
6.5 0.1
9.5 0.3
6.7 0.6
8.9 0.8
5.5 0.6
8.2 2.9
15.7 1.1
9.8 1.6
H3
H4
The physicochemical properties and drug-likeness stud-
ies of the all compounds and cisplatin were obtained and
evaluated for ADMET characteristic under Lipinski’s rule
of fve and Veber rules by using SwissADME online soft-
ware and the preADMET online server (http://preadmet.
bmdrc.org/).
H5
H6
H7
H8
H9
H10
H11
Cisplatin
Results and discussion
*SD standard deviation: measured in three independent experiments
Chemistry
According to the progressive interest on developing anti-
one-way ANOVA and Tukey’s multiple comparison tests
using GraphPad Prism 7.0 software. Values were consid-
ered signifcant when P < 0.005 (Table 1).
cancer agents based on quinazoline scafold, a series of
2-(chloromethyl)-3-phenylquinazolin-4(3H)-ones (H1–11
)
were designed and synthesized. The target compound was
designed based on the structure of the drugs in the market,
erlotinib and geftinib, the most important EGFR inhibitors
as well as some quinazolinone-based compounds reported
as anticancer agents with some modifcations (Fig. 2). In
this regard, the substituted phenyl ring was moved to the
3-position (N-3) of quinazolinone moiety and chlorine group
was located on the methyl substitution at position 2 (C-2)
to obtain an aliphatic containing chlorine group instead of
aromatic one. Moreover, to have a desired SAR and biologi-
cal behaviors, diferent aniline derivatives (D1–11) were used
to create diferent phenyl-substituted rings at N-3 position
with diversity of electronic efect of groups which could
occupy a wide range of positions. The chemical profle of
the compounds is shown in Table 2.
Docking procedure
Three-dimensional crystal structures of EGFR (PDB
ID: 1M17) were obtained from Protein Data Bank (PDB
database; http://www.rcsb.org). Water molecules and co-
crystal ligand (erlotinib) were removed, and the enzyme
was converted to PDBQT using MGLTools 1.5.6 software
[28] to prepare receptor structure. The structures of all
synthesized 2-(chloromethyl)-3-phenylquinazolin-4(3H)-
one derivatives were drawn and optimized by the Chem-
BioDraw (version 12.0) and HyperChem Professional
version 8 (Hypercube Inc., Gainesville, FL, USA); then,
geometry optimization was done by Molecular Mechanic
(MM+) following semiempirical AM1 method. The out-
puts of optimized structures were converted to PDBQT
using MGLTools. Finally, docking procedure was per-
formed by an in-house batch script (DOCK-FACE), based
on the Lamarckian genetic algorithm [29], in a parallel
mode, using all system resources, as described in our
recent studies [30, 31]. The grid points of 70, 70 and 70 Å
in all directions were centered on the ligand in the complex
with a spacing of 0.375 Å. The number of points in x-, y-
and z-directions was 20.14, 0.3 and 52.2, respectively. For
internal validation, erlotinib was excluded from the PDB
structure of receptor and was docked the same as exam-
ined ligands. Root-mean-square deviation (RMSD) value
obtained below of 2 Å showed the validity of docking
Molecular docking studies
As mentioned before, EGFR is the main target of quinazo-
line-based anticancer compounds. Molecular docking stimu-
lation was performed to evaluate the interactions and orien-
tations of all synthesized compounds toward EGFR active
site using the AutoDock Vina. Docking results contained the
estimated free binding energy (kcal/mol) of ligand–receptor
complex, and also the appropriate interactions with the main
amino acid residues at the active site of the enzymes are
shown in Table 3, Figs. 3 and 4, respectively. The binding
energy values were obtained between − 6.8 and − 8.8 kcal/
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Journal of the Iranian Chemical Society
Table 2 Physical characterization data of 2-(chloromethyl)-3-phenylquinazolin-4(3H)-one derivatives
Entry
Chemical structures
M.W
305
M.P. (found)
115
M.P. (reported)
Log P
(%)Y
H1
112–113 [33, 34]
3.96
87
H2
H3
305
173
176
174–175 [33]
180 [35]
3.96
3.56
86
83
228.7
H4
H5
H6
338
362
322
153
175
187
–
–
–
4.32
4.94
4.12
91
73
75
H7
H8
H9
H10
302
319
298
334
149
157
127
162
–
4.05
4.45
4.37
3.83
64
79
69
83
–
[35]
–
1 3

Journal of the Iranian Chemical Society
Table 2 (continued)
Entry
Chemical structures
M.W
330
M.P. (found)
174
M.P. (reported)
–
Log P
(%)Y
H11
3.15
76
Table 3 Bonding energies (kcal/mol) of compounds docked on EGFR active site
Entry
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
Erlotinib
− 5.8
Binding energy (kcal/mol)
− 8.6
− 8.1
− 8.7
− 8.4
− 8.4
− 8.8
− 8.5
− 7.2
− 8.4
− 7.1
− 6.8
mol for synthesized compounds and -5.8 kcal/mol for Erlo-
tinib. As shown in Table 2, all compounds represented better
binding energies than erlotinib as internal ligand of EGFR.
As illustrated in Fig. 3, the quinazolinone ring is involved
in hydrogen bond interaction with Met 769 at distance of
2.79 Å. Amino acids, Leu 764, Lys 721 and Thr 766 in the
side chains made hydrophobic interactions with erlotinib.
Compound H3 made three types of interactions includ-
ing hydrogen bond, Pi-cation and hydrophobic interactions
(Fig. 4). A hydrogen bond interaction existed between the
carbonyl group of H3 with Thr 766 residue in the bind-
ing site of receptor. There was also a pi-cation interaction
between 4-flourophenyl group with Lys 721 and some
hydrophobic interactions with Ala 719, Leu 820 and Lys
721. Compound H6 represented hydrogen bond interaction
Fig. 3 Interaction of erlotinib with EGFR (PDB: IM17)
Fig. 4 Interactions of H3 (a) and H6 (b) with the residues in the binding site of EGFR
1 3

Journal of the Iranian Chemical Society
via carbonyl group of quinazoline ring and hydroxyl group
of Thr 766. There were also many hydrophobic interactions
between H6 and receptor residues such as Leu 694, Ala 719,
Lys 721, Met 769 and Leu 820.
which bear di-substituted groups, the presence of electron-
withdrawing (EWD) and -donating (ED) groups together
infuenced the anticancer activities. For example, H6 showed
antiproliferative activates less than 10 μM for all investigated
cell lines which make it a promising candidate. On the other
hand, H4 and H11 derivatives which contain 3-trifuorome-
thyl and 2,4 di-methoxy groups displayed less inhibitory
activities on three studied cancer cell lines, especially for
H4, which had the least activity (Fig. 5). Overall, most of the
synthesized compounds remarkably reduce tumor growth
in SW1116 as colon cancer cell line (Fig. 6) rather than
A549 (lung carcinoma). Notably, our result indicated that H5
and H6, the most active compounds in all tested cancerous
cell lines, have also a good potential for further studies and
experiments on their anticancer properties.
Anticancer activity of synthesized compounds
Quinazoline and its derivatives are currently considered
as an important class of compounds with desirable efect
in cancer treatment. Here, we synthesized a series of
2-(chloromethyl)-3-phenylquinazolin-4(3H)-one derivatives
and evaluated their anticancer activities through inducing
cell growth inhibition in three cancerous human cell lines
with long (A549), breast (MCF-7) and colon (SW1116) ori-
gin. As summarized in Table 1, the antiproliferative activity
of compounds was varied from moderate to high inhibitory
efects even with higher cytotoxicity’s than cisplatin as a
standard drug. For A549 cell line, all compounds showed
desirable antitumor activity especially H3, H5 and H6 with
IC50 values of 5.7 0.6, 5.4 0.6 μM and 9.35 1.15 μM,
respectively, comparing to 12 1.9 μM for cisplatin. How-
ever, one-way ANOVA statistical analysis showed statisti-
cally signifcant diference only for compound H3 in com-
parison with the cisplatin. Cytotoxic activities on MCF-7
cell line represented higher cytotoxicity for synthetic com-
pounds than cisplatin, except for H3, H4 and H11. Regarding
SW1116 cell line, excluding H4 and H11, all tested com-
pounds showed higher activities than cisplatin. The difer-
ence between H1 (IC50 = 4.56 0.3 μM) as the most active
compound and cisplatin (IC50 =9.8 1.6 μM) was statisti-
cally signifcant at P<0.005.
In silico physicochemical parameters (ADME)
prediction
The physicochemical properties of all compounds and cispl-
atin were obtained by http://www.swissadme.ch/ on the basis
of Lipinski and Veber rules (Table 4). The molecular weight
(MW) of the all compounds was in the range of 228–339 (g/
mol) which stands within the accepted limit of 500 (g/mol).
The log P values of the compounds were lower than 5 which
revealed that all compounds have desired lipophilic charac-
teristic. The hydrogen bond properties (as donors or accep-
tors), total polar surface area (TPSA) and rotatable bond
number of all designed compounds are lower than accept-
able limit. Generally, these measuring data indicated that all
of the compounds followed Lipinski’s rule of fve and have
good absorption to show proper oral bioavailability.
Structure–activity relationship (SAR) of investigated
compounds revealed that in mono-substituted derivatives,
the presence of phenoxy at para-position and halogen groups
in order of Cl>F increased the anticancer activities against
SW1116 and MCF-7 cell lines. For compounds H6 to H11,
Absorbance Distribution Metabolism and Excretion
(ADME) and drug-likeness properties of designed com-
pounds and cisplatin were obtained from the preAD-
MET online server (http://preadmet.bmdrc.org/). ADME
Fig. 5 Structure–activity
relationship analysis of diferent
substitutions on phenyl ring at
position 3 of quinazolinone ring
(N-3) on their antiproliferative
efects
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Journal of the Iranian Chemical Society
Fig. 6 Antiproliferative activities of the quinazolinone-based compounds (H1-H11) and cisplatin as control against A549, MCF-7, SW1116 carci-
nomas cell lines
properties including human intestinal absorption (HIA),
in vitro Caucasian colon adenocarcinoma cell permeability
(Caco-2), skin permeability, in vitro plasma protein bind-
ing (PPB) and in vivo blood–brain Barrier penetration
(BBB) are shown in Table 5. All the compounds indicated
well human intestinal absorption (HIA), which cause good
absorption from the intestine to the bloodstream. The Caco-2
permeability parameters suggest that these compounds have
higher permeability for penetration to biological mem-
branes in comparison with cisplatin, and also most of the
compounds bind moderately to plasma protein; therefore,
they can difuse or transport across the cell membranes. On
the other hand, compounds H5, H7, H9 and H11 have lower
blood–brain barrier percentage compared to cisplatin and
probably will not cause neurotoxicity efect.
Conclusion
A series of 2-(chloromethyl)-3-phenylquinazolin-4(3H)-
one derivatives were successfully synthesized and char-
acterized by melting points, H-NMR, 13CNMR, IR and
1
mass spectroscopy. The in vitro cytotoxic activities of each
compound against three human cancerous cell lines (non-
small cell lung cancer, breast cancer and colon cancer) were
evaluated that almost all synthesized compounds had higher
antitumor activities than cisplatin, the standard anticancer
drug, except for H4 and H11. Among them, H3, H5 and H6
exhibited higher potencies in inhibiting cell proliferation.
The molecular docking studies of these compounds on
EGFR confrmed that there is a good correlation between the
docking studies and the antitumor activities, especially for
1 3

Journal of the Iranian Chemical Society
Table 4 Physicochemical
properties of the synthesized
compounds
Entry
MWa
Log Pb
HBDc
HBAd
TPSA (A2)e
n-RBf
Lipinski
violation
H1
305.16
305.16
288.70
338.71
362.81
323.15
302.73
319.19
298.77
335.18
330.77
298.03
≤500
3.96
3.96
3.56
4.32
4.94
4.12
4.05
4.45
4.45
4.37
3.83
3.15
≤5
0
0
0
0
0
0
0
0
0
0
0
0
≤5
2
2
2
3
3
3
3
2
2
3
4
4
≤10
34.89
34.89
34.89
34.89
44.12
34.89
34.89
34.89
34.89
44.12
53.35
52.04
≤140
2
2
3
5
4
2
2
2
2
3
4
0
≤10
0
0
0
0
0
0
0
0
0
0
0
0
≤1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
Cisplatin
Rule of Lipinski
aMolecular weight (MW)
bLogarithm of partition coefcient between n-octanol and water (Log P)
cNumber of hydrogen bond donors (HBD)
dNumber of hydrogen bond acceptors (HBA)
eTopological polar surface area(TPSA)
fNumber of rotatable bonds (nRB)
rules. Accordingly. H6 might be considered as a potential
anticancer agent for further in vitro and in vivo studies.
Table 5 In silico ADME of the all compounds
Supplementary Information The online version contains supplemen-
Entry
Absorption
Distribution
%BBBb
tary material available at https://doi.org/10.1007/s13738-021-02168-1.
% HIAa In vitro Caco-2 % In vitro
Acknowledgements Financial support from the Shiraz University of
Medical Sciences with Grant numbers of 98-01-36-21224 and 99-01-
16-23354 is gratefully acknowledged.
cell permeabil- plasma protein
ity (nm/s)
bonding
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
99.02
99.02
98.88
98.92
97.99
99.02
98.91
99.09
98.94
97.8
33.49
32.95
24.29
27.68
25.94
35.65
24.94
34.68
24.25
34.15
24.08
19.88
91.16
91.77
87.7
2.1
1.25
2.4
1.71
0.07
1.6
0.11
1.6
0.16
2.8
0.03
0.38
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91.55
92.55
91.32
91.04
90.98
91.63
85.98
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