Synthesis of some quinazolinones inspired from the natural alkaloid L-norephedrine as EGFR inhibitors and radiosensitizers

Article abstract of DOI:10.1080/14756366.2020.1854243

A set of quinazolinones synthesized by the aid of L-norephedrine was assembled to generate novel analogues as potential anticancer and radiosensitizing agents. The new compounds were evaluated for their cytotoxic activity against MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer cell lines and EGFR inhibitory activity. The most active compounds 5 and 6 were screened against MCF-10A normal cell line and displayed lower toxic effects. They proved their relative safety with high selectivity towards MDA-MB-231 breast cancer cell line. Measurement of the radiosensitizing activity for 5 and 6 revealed that they could sensitize the tumour cells after being exposed to a single dose of 8 Gy gamma radiation. Compound 5 was able to induce apoptosis and arrest the cell cycle at the G2-M phase. Molecular docking of 5 and 6 in the active site of EGFR was performed to gain insight into the binding interactions with the key amino acids.

Full text of DOI:10.1080/14756366.2020.1854243

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis of some quinazolinones inspired from
the natural alkaloid L-norephedrine as EGFR
inhibitors and radiosensitizers
Mostafa M. Ghorab, Maged S. Abdel-Kader, Ali S. Alqahtani & Aiten M.
Soliman
To cite this article: Mostafa M. Ghorab, Maged S. Abdel-Kader, Ali S. Alqahtani & Aiten M.
Soliman (2021) Synthesis of some quinazolinones inspired from the natural alkaloid L-norephedrine
as EGFR inhibitors and radiosensitizers, Journal of Enzyme Inhibition and Medicinal Chemistry,
36:1, 218-237, DOI: 10.1080/14756366.2020.1854243
To link to this article: https://doi.org/10.1080/14756366.2020.1854243
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2020 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
021, VOL. 36, NO. 1, 218–237
2
https://doi.org/10.1080/14756366.2020.1854243
RESEARCH PAPER
Synthesis of some quinazolinones inspired from the natural alkaloid
L-norephedrine as EGFR inhibitors and radiosensitizers
a
b,c
d,e
a
Mostafa M. Ghorab
, Maged S. Abdel-Kader , Ali S. Alqahtani and Aiten M. Soliman
a
Department of Drug Radiation Research, National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority
b
(
EAEA), Cairo, Egypt; Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-Kharj, Saudi Arabia;
Department of Pharmacognosy, College of Pharmacy, Alexandria University, Alexandria, Egypt; Department of Pharmacognosy, College of
c
d
e
Pharmacy, King Saud University, Riyadh, Saudi Arabia; Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of
Pharmacy, King Saud University, Riyadh, Saudi Arabia
ABSTRACT
ARTICLE HISTORY
A set of quinazolinones synthesized by the aid of L-norephedrine was assembled to generate novel ana-
logues as potential anticancer and radiosensitizing agents. The new compounds were evaluated for their
cytotoxic activity against MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer cell lines and EGFR inhibitory
activity. The most active compounds 5 and 6 were screened against MCF-10A normal cell line and dis-
played lower toxic effects. They proved their relative safety with high selectivity towards MDA-MB-231
breast cancer cell line. Measurement of the radiosensitizing activity for 5 and 6 revealed that they could
sensitize the tumour cells after being exposed to a single dose of 8 Gy gamma radiation. Compound 5
was able to induce apoptosis and arrest the cell cycle at the G2-M phase. Molecular docking of 5 and 6 in
the active site of EGFR was performed to gain insight into the binding interactions with the key amino
acids.
Received 16 September 2020
Revised 3 November 2020
Accepted 17 November 2020
KEYWORDS
Quinazolinone; cytotoxicity;
EGFR; anticancer; docking
GRAPHICAL ABSTRACT
1
. Introduction
inhibition of EGFR is classified as targeted therapy as it aims at
the differences between cancer and normal cells and is character-
ized by its high selectivity and lowered side effects.
Cancer is characterized by the disturbance of normal cellular proc-
esses required for cell growth, division and differentiation
Surgery, radiotherapy and chemotherapy, including immunother-
apy, targeted and combined therapy, are different strategies advo-
cated for cancer treatment
Protein kinases (PKs) play a pivotal role in cell proliferation by
controlling signal transduction through the phosphorylation of dif-
ferent amino acid residues, namely tyrosine, threonine and serine .
1
–3
.
Quinazolines are fused heterocyclic ring systems known for
1
2–15
their variable biological activity
. They are well known for their
4–6
inhibitory activity towards various protein kinase enzymes and
.
1
6
their anticancer activity . For example, lapatinib, a dual reversible
EGFR and HER2 inhibitor. Also, gefitinib and erlotinib are revers-
ible EGFR inhibitors; they are examples of FDA approved small
7
1
7
molecules TK inhibitors . Methaqualone, a potent hypnotic, was
considered as an important landmark in synthetic anticonvul-
Tyrosine kinases (TKs) are divided into receptor tyrosine kinases
(RTKs) and non-receptor tyrosine kinases (NRTKs) in human gen-
1
8
sants . The 3-[b-keto-gamma-(3-hydroxy-2-piperidyl)-propyl]-4-qui-
nazolone (A) was the first isolated natural quinazolinone alkaloid
ome. RTKs are vital components of cellular signaling pathways
that are active during embryonic development and adult homeo-
stasis. Due to their role as growth factor receptors, many RTKs
have been involved in the onset or progression of various cancers,
either by mutations or receptor/ligand overexpression; thus, they
are considered attractive candidates for therapeutic interven-
tion . An example of RTK family members is epidermal growth
factor receptor (EGFR). EGFR is a member of the ErbB receptor
family and plays an essential role in cell signaling. Signaling is ini- severe side effects, including fatality . In addition to medicinal
tiated by binding ligands to the extracellular domain of the EGFR, use, the properties of this alkaloid have attracted considerable
activating kinases and promoting cancer cell survival, invasiveness attention in natural product chemistry field that leads to its use as
1
9
known by its antimalarial activity . The quinazolinone derivatives
(
B) and benzo[g]quinazolinone (C) were reported to possess
2
0,21
potent EGFR and HER2 inhibitory activity
(Figure 1). On the
other hand, the Ephedra alkaloid, Norephedrine (NE) is a stereoiso-
mer of phenylpropanolamine that is naturally occurring sympatho-
7
,8
2
2
mimetic . Investigation revealed that long-term use of NE caused
2
3
9
,10
and drug resistance . EGFR has a critical role in regulating sev- a starting material in the preparation of chiral ligands for asym-
eral cellular functions such as cell growth, proliferation, differenti- metric catalytic synthesis
2
4,25
.
ation and apoptosis, leading to the development of several types
In continuation of our studies aiming to find new leads with
of solid tumors . EGFR (HER-1) and ERB-B2 (HER-2) are character- potential anticancer activities, various substituted quinazolinones
ized in solid tumors as breast, ovary, lung and others. The have been designed to accommodate different electronic natures
1
1
CONTACT Mostafa M. Ghorab
NCRRT), Egyptian Atomic Energy Authority (EAEA), Nasr City P.O. Box 29, Cairo 11765, Egypt
Supplemental data for this article can be accessed here.
mmsghorab@yahoo.com
Department of Drug Radiation Research, National Center for Radiation Research and Technology
(
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
219
Figure 1. Quinazoline-based scaffolds used to design our target compounds.
as heterocycles representing the primary scaffold in many cyto- ol) (0.151 g, 0.001 mol) 2 in NMR tube in CDCl and measured
3
1
13
toxic agents hoping to develop potent and safe anticancer agents immediately for H and C NMR to give 3. When the reaction
and EGFR inhibitors. All the synthesized compounds were was carried out in the presence of chloroform containing a cata-
screened against MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer lytic amount of triethylamine the reaction gave 4 instead of 3 at
cell lines and the most potent compounds were evaluated against room temperature. The product 4 was crystallized from ethanol.
MCF-10A normal cells to determine the selectivity of the com- Derivative 3 was rapidly converted to 4 at room temperature.
1
pounds on the different cell lines. Also, the in vitro EGFR inhibitory
3: H NMR (500 MHz, CDCl
3
): 0.99 (d, J ¼ 6.0 Hz, 3H, CH
), 4.81 (bs, 1H,
potent compounds on cell cycle progression and the radiosensitiz- N–CH), 5.13 (bs, 1H, O–CH), 6.64 (d, J ¼ 6.8 Hz, 1H, OH), 7.05–7.93
3
of L-
activity of the compounds was measured. The effect of the most norephedrine), 2.87 (bs, 1H, NH), 3.83 (s, 3H, O–CH
3
1
3
ing activity were evaluated. Docking studies were carried out to (m, 9 aromatic), 10.31 (bs, 1H, NH). C NMR (126 MHz, CDCl
3
):
confirm the possible mode of action of the promis- 13.30 (CH ), 55.76 (O–CH ), 75.20 (N–CH), 77.33 (O–CH), 118.80,
3
3
ing compounds.
122.74, 123.69, 125.06, 126.92 (2), 128.35 (2), 131.44, 133.31,
þ
1
40.65 (2), 167.96, 179.68. MS m/z (%): 344 (M ) (1.96), 179 (100).
ꢀ
ꢁ1
4
: Yield, 83%; m.p. 80.3 C. IR (KBr, cm ): 3455 (OH), 3244
1
2
. Materials and methods
(NH), 3088 (arom.), 2970, 2865 (aliph.), 1691 (CO), 1277 (CS). H
NMR (500 MHz, DMSO-d ): d 1.60 (d, J ¼ 6.0 Hz, 3H, CH of L-nore-
6
3
2.1. Chemistry
phedrine), 5.33 (bs, 1H, N–CH), 5.66 (bs, 1H, O–CH), 6.11 (bs, 1H,
Melting points were determined uncorrected by a Gallen Kamp
melting point apparatus (Sanyo Gallen Kamp, UK). Precoated silica
gel plates (Kieselgel 0.25mm, 60 F254, Merck, Germany) were used
for TLC with solvent system of chloroform/methanol (8:2), spots
were detected by UV light. IR spectra (KBr discs) were recorded
NH), 7.06–7.30 (m, 7 aromatic), 7.63 (t, J ¼ 6.0 Hz, 1H), 7.88 (d,
13
J ¼ 7.0 Hz, 1H), 12.30 (s, 1H, OH). C NMR (126 MHz, DMSO-d
6
): d
1
1
1
3.93 (CH ), 61.15 (N–CH), 73.84 (O–CH), 115.20, 116.04, 124.39,
3
26.74 (2), 126.87, 127.41, 128.12 (2), 135.33, 138.51, 142.37,
þ
59.57, 176.01. MS m/z (%): 312 (M ) (10), 78 (100). Anal. Calcd.
1
13
using FT-IR spectrophotometer (Perkin Elmer, USA). H, C NMR
and 2D NMR experiments were scanned on an NMR spectropho-
16 2 2
For C17H N O S (312): C, 65.36; H, 5.16; N, 8.97. Found: C, 65.57;
H, 5.41; N, 9.19.
1
tometer (Bruker AXS Inc., Switzerland), operating at 500 MHz for H
1
3
and 125.76MHz for C. Chemical shifts are expressed in d-values
ppm) relative to TMS as an internal standard, using DMSO-d and 2.1.2. 3-(1-Hydroxy-1-phenylpropan-2-yl)-2-(methylthio)quinazolin-
(
6
3
CDCl as solvents. EIMS were measured using Shimadzu-GC/MS. 4(3H)-one (5)
Elemental analyses were performed on a model 2400 CHNSO ana- A mixture of 4 (0.312 g, 0.001 mol) and methyl iodide (0.141 g,
lyser (Perkin Elmer, USA). All the values were within ±0.4% of the 0.001 mol) in dry acetone (30 mL) containing K CO was refluxed
2
3
theoretical values. The X-ray data were collected at T ¼ 298 K on for 12 h. The obtained solid was crystallized from ethanol to
Enraf Nonius 590 Kappa CCD single crystal diffractometer equipped give 5.
ꢀ
ꢁ1
with graphite monochromated Mo Ka (k¼0.71073 Å) radiation using
w–x scan technique. All reagents used were of AR grade.
5: Yield, 78%; m.p. 121.5 C. IR (KBr, cm ): 3405 (OH), 3055
1
(arom.), 2966, 2871 (aliph.), 1683 (CO), 1612 (CN). H NMR
(
500 MHz, DMSO-d
6
): d 1.71 (d, J ¼ 6.5 Hz, 3H, CH
3
of L-norephe-
drine), 2.47 (s, 3H, S–CH ), 4.45 (bt, 1H, N–CH), 5.57 (bs, 1H, O–CH),
3
2
.1.1. Methyl 2-(3-(1-hydroxy-1-phenylpropan-2-yl)thioureido)ben-
5
.87 (bs, 1H, OH), 7.10–7.38 (m, 7 aromatic), 7.69 (t, J ¼ 6.5 Hz, 1H),
1
3
zoate (3) & 3-(1-hydroxy-1-phenylpropan-2-yl)-2-thioxo-2,3-dihy- 8.09 (d, J ¼ 7.0 Hz, 1H). C NMR (126 MHz, DMSO-d ): d 14.62
6
droquinazolin-4(1H)-one (4)
3 3
(CH ), 15.37 (S–CH ), 61.98 (N–CH), 72.80 (O–CH), 119.34, 125.51,
Methyl 2-isothiocyanatobenzoate
allowed to react with L-norephedrine (2-amino-1-phenylpropan-1- 146.04, 157.15, 161.09. MS m/z (%): 296 (M -2CH
1
(0.193 g, 0.001 mol) was 125.76, 126.07, 126.36 (2), 127.42, 127.58 (2), 134.55, 142.16,
þ
3
) (12), 180 (100).

2
20
M. M. GHORAB ET AL.
Anal. Calcd. For C18
Found: C, 66.49; H, 5.88; N, 8.87.
H
18
N
2
O
2
S (326): C, 66.23; H, 5.56; N, 8.58 (500 MHz, DMSO-d
6
): d 1.72 (d, J ¼ 6.5 Hz, 3H, CH
3
of L-norephe-
drine), 4.24 (t, J ¼ 7.0 Hz, 1H, N–CH), 5.86 (d, J ¼ 7.5 Hz, 1H, S–CH),
1
3
7
.10–7.85 (9 aromatic), 8.12 (d, 1H, NH). C NMR (126 MHz,
DMSO-d ): d 14.75 (CH ), 45.98 (N–CH), 72.69 (S–CH), 119.58,
25.83, 126.16, 126.30 (2), 126.50, 127.54, 127.69 (2), 134.84,
41.95, 145.64, 152.85, 161.10. MS m/z (%): 309 (M ) (17), 79 (100).
OS (309): C, 66.00; H, 4.89; N, 13.58.
6
3
2
.1.3. 3-Amino-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (6)
1
1
The method for the synthesis of compound 6 was reported by El-
þ
2
6
Hiti et al .
: Yield, 89%; m.p. 261.2 C. IR (KBr, cm ): 3356, 3280, 3176
15 3
Anal. Calcd. For C17H N
ꢀ
ꢁ1
6
Found: C, 66.31; H, 5.16; N, 13.89.
1
(
NH , NH), 3096 (arom.), 1696 (CO), 1283 (CS). H NMR (500 MHz,
ꢀ
ꢁ1
2
1
1: Yield, 23%; m.p. 79.5 C. IR (KBr, cm ): 3460 (OH), 3391,
DMSO-d ): d 6.39 (s, 2H, NH ), 7.37 (t, J ¼ 7.0, 1H), 7.42 (d,
6
2
3
1
312, 3215 (NH
608 (CN). H NMR (500 MHz, DMSO-d
norephedrine), 3.43 (s, 1H, NH), 4.29 (bs, 1H, N–CH), 4.91 (bs, 1H,
O–CH), 5.57 (s, 2H, NH ), 5.73 (s, 1H, OH), 7.13–7.45 (8 aromatic),
2
, NH), 3072 (arom.), 2946, 2815 (aliph.), 1678 (CO),
): d 0.96 (bs, 3H, CH of L-
J ¼ 7.3 Hz, 1H), 7.75 (t, J ¼ 7.0, 1H), 7.99 (t, J ¼ 7.0, 1H), 12.30 (s, 1H,
1
1
3
6
3
NH). C NMR (126 MHz, DMSO-d
6
): d 114.70, 115.78, 124.39,
þ
1
1
2
26.67, 134.85, 138.21, 155.41, 169.27. MS m/z (%): 193 (M ) (95),
62 (100). Anal. Calcd. For C H N OS (193): C, 49.73; H, 3.65; N,
1.75. Found: 49.48; H, 3.31; N, 21.48.
2
8
7
3
13
7
1
1
1
.93 (d, J ¼ 7.2 Hz, 1H, aromatic). C NMR (126 MHz, DMSO-d
6
): d
3.44 (CH ), 51.74 (N–CH), 73.43 (O–CH), 116.47, 121.40, 124.49,
3
25.97, 126.48 (2), 126.59, 127.29, 127.91 (2), 133.94, 143.01,
50.25, 161.10. MS m/z (%): 310 (M ) (21) 78 (100). Anal. Calcd.
þ
2
.1.4. 3-Amino-2-(methylthio)quinazolin-4(3H)-one (7)
For C H N O (310): C, 65.79; H, 5.85; N, 18.05. Found: C, 66.15;
A mixture of 6 (0.193 g, 0.001 mol) and methyl iodide (0.141 g,
.001 mol) was refluxed in dry acetone containing K CO for 12 h.
17 18 4 2
H, 6.11; N, 18.39.
0
2
3
The reaction mixture was filtered and crystallized from ethanol to
give 7.
ꢀ
ꢁ1
2.1.7. 3-Amino-2-hydroxyquinazolin-4(3H)-one (12), 3-amino-2-
ethoxyquinazolin-4(3H)-one (13) and [1,2,4,5]tetrazino[3,2-b:6,5-
7: Yield, 81%; m.p. 178.9 C. IR (KBr, cm ): 3431, 3257 (NH
2
),
1
3
100 (arom.), 2919, 2866 (aliph.), 1689 (CO), 1618 (C¼N). H NMR
(
500 MHz, DMSO-d ): d 2.44 (s, 3H, S–CH ), 5.77 (s, 2H, NH ), b’]diquinazoline-8,16(6H,14H)-dione (14)
6 3 2
13
7
1
.41–8.07 (m, 4H, Ar–H). C NMR (126 MHz, DMSO-d ): d 14.09, A mixture of 7 (0.207 g, 0.001 mol) and 2 (0.151 g, 0.001 mol) was
18.75, 125.29, 125.97, 126.07, 134.27, 147.03, 160.49, 160.94. MS refluxed in ethanol 95% (30 mL) containing K
6
CO (0.138 g,
3
2
þ
m/z (%): 207 (M ) (26), 58 (100). Anal. Calcd. For C
9
H
9
N
3
OS (207): 0.001 mol) for 12 h. The reaction was monitored by TLC and indi-
C, 52.16; H, 4.38; N, 20.27. Found: C, 52.02; H, 4.16; N, 20.01.
cated the presence of two products 12 and 13. The products
were separated by silica gel column chromatography (45 ꢂ 2 i.d.
cm, 30 gm) eluting with chloroform, followed by chloroform/
methanol mixtures in a gradient system. Fractions 12–15 eluted
with 2% methanol in chloroform afforded 13 (117 mg) after crys-
tallization from methanol. Fractions 23–26 eluted with 5% metha-
nol in chloroform afforded 12 (43 mg) after crystallization from
methanol. When the reaction was repeated but in the presence of
DMF instead of ethanol, dimer 14 was formed instead of 11. The
products obtained were crystallized from dioxane.
2
.1.5. 3-Methyl-2-phenyl-2H-thiazolo[2,3-b]quinazolin-5(3H)-one (8)
27
Compound 8 was reported by Ghorab et al .
2
.1.6. 3-Methyl-2-phenyl-3,4-dihydro-[1,3,4]oxadiazino[2,3-b]quina-
zolin-6(2H)-one (9), 3-methyl-2-phenyl-3,4-dihydro-[1,3,4]thiadia-
zino[2,3-b]quinazolin-6(2H)-one (10) and 3-amino-2-(1-hydroxy-1-
phenylpropan-2-ylamino)quinazolin-4(3H)-one (11)
ꢀ
ꢁ1
1
2: Yield, 24%; m.p. 294.1 C. IR (KBr, cm ): 3488 (OH), 3212,
To a solution of 1 (0.193 g, 0.001 mol) in ethanol (25 mL) with 2
1
3
152 (NH ), 3055 (arom.), 1680 (CO), 1606 (CN). H NMR (500 MHz,
2
(0.151 g, 0.001 mol), hydrazine hydrate (0.05 g, 0.001 mol) was
6 2
DMSO-d ): d 5.50 (s, 2H, NH ), 7.19–7.24 (m, 2H), 7.30–7.65 (m,
added and refluxed for 20 h. The progress of the reaction was
monitored by TLC that indicates the presence of three products.
The mixture was filtered to give three compounds 9, 10 and 11.
The mixture was separated by silica gel column chromatography
13
1
(
1
H), 7.94 (dd, J ¼ 1.2, 8.0 Hz, 1H), 11.62 (s, 1H, OH). C NMR
126 MHz, DMSO-d ): d 113.87, 115.69, 123.02, 127.34, 134.93,
38.61, 148.93, 159.61. MS m/z (%): 177 (M ) (36), 118 (100). Anal.
6
þ
Calcd. For C
5
8
H
7
N
3
O
2
(177): C, 54.24; H, 3.98; N, 23.72. Found: C,
4.52; H, 4.21; N, 24.01.
3: Yield, 57%; m.p. 102.8 C. IR (KBr, cm ): 3220, 3182 (NH ),
(
45 ꢂ 2 i.d. cm, 30 gm) eluting with chloroform, followed by
chloroform/methanol mixtures in a gradient system. Fractions 4–7
eluted with chloroform afforded 9 (103 mg) after crystallization
from methanol. Fractions 10–12 eluted with chloroform afforded
ꢀ
ꢁ1
1
2
1
3
099 (arom.), 2918, 2844 (aliph.), 1679 (CO), 1920 (CN). H NMR
): d 1.39 (t, J ¼ 7.0, 3H, CH ), 4.49 (q, J ¼ 7.0,
H, CH ), 5.72 (s, 2H, NH ), 7.33 (t, J ¼ 7.3, 1H), 7.44 (d, J ¼ 8.0 Hz,
(500 MHz, DMSO-d
6
3
10 (68 mg) after crystallization from methanol. Fractions 21– 24
2
1
2
2
eluted with 5% methanol in chloroform afforded 11 (71 mg) after
1
3
H), 7.69 (m, 1H), 8.01 (dd, J ¼ 1.0, 8.0 Hz, 1H). C NMR (126 MHz,
crystallization from methanol.
ꢀ
ꢁ1
DMSO-d
6
): d 14.61 (CH
3
), 64.79 (CH
2
), 118.14, 124.56, 125.87,
9: Yield, 35%; m.p. 147.5 C. IR (KBr, cm ): 3149 (NH), 3098
þ
1
126.59, 134.44, 146.12, 151.56, 160.30. MS m/z (%): 205 (M ) (32),
(
(
arom.), 2946, 2907 (aliph.), 1685 (CO), 1602 (CN). H NMR
1
28 (100). Anal. Calcd. For C H N O (205): C, 58.53; H, 5.40; N,
10 11 3 2
500 MHz, DMSO-d ): d 0.95 (d, J ¼ 6.5 Hz, 3H, CH of L-norephe-
6
3
2
0.48. Found: C, 58.88; H, 5.76; N, 20.76.
drine), 5.18 (t, J ¼ 7.0 Hz, 1H, N–CH), 6.18 (d, J ¼ 8.0 Hz, 1H, O–CH),
ꢀ
ꢁ1
13
1
4: Yield, 76%; m.p. 201.4 C. IR (KBr, cm ): 3217, 3176 (NH),
7
.37–7.78 (9 aromatic), 8.01 (d, 1H, NH). C NMR (126 MHz,
DMSO-d ): d 13.96 (CH ), 53.96 (N–CH), 81.86 (O–CH), 118.83,
24.48, 125.84, 126.14, 126.32 (2), 128.52 (2), 128.69, 133.66,
1
3
6
075 (arom.), 1696 (CO), 1618 (CN). H NMR (500 MHz, DMSO-d ):
6
3
d 3.42 (bs, 2H, 2NH), 7.38 (t, J ¼ 7.5 Hz, 2H), 7.50 (d, J ¼ 7.5 Hz, 2H),
1
1
þ
13
34.72, 148.76, 154.84, 159.86. MS m/z (%): 293 (M ) (1.95), 64 7.70 (t, J ¼ 7.5 Hz, 2H), 8.02 (d, J ¼ 7.5 Hz, 2H). C NMR (126 MHz,
(100). Anal. Calcd. For C17
15
H N
O
3 2
(293): C, 69.61; H, 5.15; N, 14.33. DMSO-d
6
): d 120.34 (2), 125.93 (2), 126.40 (2), 128.73 (2), 134.94
(2), 148.79 (2), 156.84 (2), 161.67 (2). MS m/z (%): 318 (M ) (28),
þ
Found: C, 69.40; H, 4.86; N, 14.05.
ꢀ
ꢁ1
1
0: Yield, 22%; m.p. 219.9 C. IR (KBr, cm ): 3210 (NH), 3100 158 (100). Anal. Calcd. For C16
H
10 6
N O
2
(318): C, 60.38; H, 3.17; N,
1
(arom.), 2939, 2810 (aliph.), 1678 (CO), 1610 (CN). H NMR 26.40. Found: C, 60.07; H, 2.82; N, 26.07.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
ꢁ1
221
ꢀ
15: Yield, 40%; m.p. 136.6 C. IR (KBr, cm ): 3048 (arom.),
2
(
.1.8.
3-Methyl-2-phenyl-2H-oxazolo[2,3-b]quinazolin-5(3H)-one
1
15), 2,4-dimethyl-1,3-diphenyl-3,4-dihydro-2a,4a,9b-triazapenta- 2970, 2816 (aliph.), 1690 (CO), 1621 (CN). H NMR (500 MHz,
1
DMSO-d
6
): d 0.95 (d, J ¼ 6.7 Hz, 3H, CH
3
of L-norephedrine), 5.17
leno[1,6-ab]naphthalen-5(2a H)-one
(16),
8,18-dimethyl-7,17-
(
(
(
1
(
p, J ¼ 7.6 Hz, 1H, N–CH), 6.18 (d, J ¼ 7.6 Hz, 1H, O–CH), 7.35–8.08
diphenyl-7,8,17,18-tetrahydro-[1,6,3,8]dioxadiazecino[2,3-b:7,8-
b’]diquinazoline-10,20-dione (17) and 3-(1-hydroxy-1-phenylpro-
pan-2-yl)-2-(1-hydroxy-1-phenylpropan-2-ylamino)quinazolin-
1
3
m, 9H). C NMR (126 MHz, DMSO-d ): d 14.45 (CH ), 54.49
6
3
N–CH), 82.37 (O–CH), 119.33, 124.90, 126.31, 126.61, 126.80 (2),
28.90 (2), 129.15, 134.14, 135.12, 149.24, 155.30, 160.34. MS m/z
4
(3H)-one (18)
To a solution of 5 (0.326 g, 0.001 mol) in DMF (20 mL) containing
CO (0.138 g, 0.001 mol), L-norephedrine 2 (0.151 g, 0.001 mol)
þ
%): 278 (M ) (44), 77 (100). Anal. Calcd. For C H N O (278): C,
1
7 14 2 2
7
3.37; H, 5.07; N, 10.07. Found: C, 73.65; H, 5.32; N, 10.32.
K
2
3
ꢀ
ꢁ1
1
6: Yield, 12%; m.p. >350 C. IR (KBr, cm ): 3077 (arom.),
was added and refluxed for 10 h. The reaction mixture progress
was monitored by TLC. It showed the presence of four products
1
matography (45 ꢂ 2 i.d. cm, 40 gm) eluting with chloroform, fol-
lowed by chloroform/methanol mixtures in a gradient system.
Fractions 3–8 eluted with chloroform afforded 15 (111 mg) after
crystallization from methanol. Fractions 11–12 eluted with chloro-
form afforded 17 (56 mg) after crystallization from methanol.
Fractions 16–18 eluted with 2% methanol in chloroform afforded
1
2
(
927, 2846 (aliph.), 1693 (CO). H NMR (500 MHz, DMSO-d ): d 1.23
6
d, J ¼ 11.5 Hz, 3H, CH of L-norephedrine), 1.24 (d, J ¼ 12.0 Hz, 3H,
3
5, 16, 17 and 18 that were separated by silica gel column chro-
CH of L-norephedrine), 4.73 (d, J ¼ 11.0 Hz, 1H), 4.79 (d,
3
J ¼ 12.0 Hz, 1H), 5.33 (p, J ¼ 7.0, 1H), 5.46 (p, J ¼ 7.2, 1H), 7.21–8.00
13
(
1
(
1
1
m, 14H, aromatic). C NMR (126 MHz, DMSO-d ): d 16.49 (CH ),
6 3
6.74 (CH ), 51.18 (N–CH), 52.64 (N–CH), 52.99 (N–CH), 53.43
3
N–CH), 113.76, 114.90, 115.49, 123.07, 127.69, 127.90, 128.41 (2),
28.74 (2), 129.17, 135.55, 135.63, 139.89, 140.12, 140.84, 150.24,
þ
51.33, 162.77, 163.12. MS m/z (%): 394 (M ) (14), 235 (100). Anal.
1
6 (47 mg) after crystallization from methanol. Fractions 27–29
24 3
Calcd. For C26H N O (394): C, 79.16; H, 6.13; N, 10.65. Found: C,
eluted with 5% methanol in chloroform afforded 18 (34 mg) after
crystallization from methanol.
7
9.51; H, 6.35; N, 10.89.
Scheme 1. Synthesis of derivatives 3–7.

2
22
M. M. GHORAB ET AL.
ꢁ
1
1
7: Yield, 10%; semisolid. IR (KBr, cm ): 3101 (arom.), 2933, Collection. The 96-well plate was incubated for 24 h before the
1
2
818 (aliph.), 1690 (2CO), 1622 (2CN). H NMR (500 MHz, DMSO- MTT assay. The cell layer was washed with 0.25% (w/v) Trypsin,
d
6
): d 0.60 (d, J ¼ 6.2 Hz, 6H, 2CH
3
of L-norephedrine), 4.16 (P, 0.53 mM EDTA solution. Cells were cultured using DMEM supple-
J ¼ 1.0 Hz, 2H, 2 (N–CH)), 5.68 (d, J ¼ 8.5 Hz, 2H, 2 (O–CH)), mented with 10% foetal bovine serum, 10 mg/mL insulin and 1%
1
3
penicillin–streptomycin. Reconstituted MTT (10%) was added and
incubated for 2 h. Formazan crystals were dissolved by the MTT
solubilizing solution after incubation. Absorbance was measured
7
5
(
(
2
.12–7.87 (m, 18H). C NMR (126 MHz, DMSO-d ): d 13.74 (2),
3.02 (2) (N–CH), 79.94 (2) (O–CH), 113.57 (2), 123.47 (2), 126.53
6
2), 127.99 (2), 128.58 (4),128.81 (4), 132.72 (2), 136.34 (2), 139.62
2
8
þ
at a wavelength of 570 nm . IC was estimated according to the
2), 149.71 (2), 158.88 (2), 161.80 (2). MS m/z (%): 556 (M ) (13),
50
equation of Boltzmann sigmoidal concentration–response curve
and compared to erlotinib and staurosporine.
78 (100). Anal. Calcd. For C H N O (556): C, 73.37; H, 5.07; N,
34 28 4 4
1
0.07. Found: C, 73.08; H, 4.89; N, 9.11.
ꢀ
ꢁ1
1
8: Yield, 8%; m.p. 88.8 C. IR (KBr, cm ): 3450 (2OH),
3
321(NH), 3079 (arom.), 2976, 2823 (aliph.), 1680 (CO), 1618 (CN).
1
H NMR (500 MHz, DMSO-d
6
): d 1.58 (d, J ¼ 6.6 Hz, 6H, 2 (CH
3
) of
L-norephedrine), 4.15, 4.40 (bs, 1NH), 5.10 (bs, J ¼ 11.5 Hz, 2H, 2
(
N–CH)), 5.24 (m, 2H, 2 (O–CH)), 5.62 (d, J ¼ 5.3 Hz, 2H, 2 (OH)),
13
7
1
1
1
6
.01–7.55 (m, 14H, aromatic). C NMR (126 MHz, DMSO-d ): d
5.31 (CH ) (2), 56.53 (N–CH) (2), 74.05 (2), 115.14, 122.80 (2),
3
26.96, 127.63, 128.03, 128.30 (4), 128.65 (4), 135.28, 139.58 (2),
þ
43.50, 150.47, 162.45. MS m/z: 430 (M þ 1) (100). Anal. Calcd.
27 3 3
For C26H N O (429): C, 72.71; H, 6.34; N, 9.78. Found: C, 72.46; H,
6
.08; N, 9.40.
2.2. Biological evaluation
2
.2.1. MTT assay
MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer cell lines and MCF-
0A normal cells were obtained from American Type Culture Figure 2. X-ray crystallographic structure of compound 8.
1
Scheme 2. Synthesis of compound 8.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
37
223
1
2
.2.2. EGFR assay
Authority, using Gamma cell-40 ( Cs) source. The promising
EGFR kinase kit (0.192 mg/mL) was obtained from Invitrogen. An compounds 5 and 6 were selected to be re-evaluated for in vitro
ATP solution and a kinase/peptide mixture were developed just cytotoxic activity after the cells containing the compounds were
before use. The solution on the plate was mixed carefully and gamma-irradiated at a dose level of 8 Gy with a dose rate of
ꢀ
incubated for 1 h at 25 C. Then, 5 mL of the prepared solution 0.758 rad/s for 17.59 min. Cytotoxicity was measured two days
was added to each well. The plate was incubated for 1 h and after irradiation. The IC50 of the tested compounds is calculated
determined by an ELISA Reader (PerkinElmer, USA). Curve fitting using GraphPad Prism 5.
using Graph Pad Prism 5 was constructed. Each experiment was
repeated three times. IC50 was represented as means ± SE.
2.2.4. Cell cycle analysis
5
The MDA-MB-231 cells (10 /well) were incubated with compound
2
.2.3. Radiosensitizing evaluation
5 at its IC50. After 24 h, the cells were washed twice with PBS,
Irradiation was performed at the National Centre for Radiation then collected and fixed with ice-cold ethanol 70% (v/v). The cells
Research and Technology (NCRRT), Egyptian Atomic Energy were re-suspended with 0.1 mg/mL RNase, stained with 40 mg/mL
Scheme 3. Synthesis of compounds 9–11.

2
24
M. M. GHORAB ET AL.
PI and examined using flow cytometry (FACScalibur- 3. Results and discussion
Becton Dickinson).
3.1. Chemistry
The behaviour of the methyl 2-isothiocyanatobenzoate 1 towards
the natural alkaloid L-norephedrine 2 was studied. When the iso-
thiocyanate 1 reacted with 2 in chloroform containing a catalytic
2
.2.5. Apoptotic assay
Cells were prepared as previously mentioned. Treatment of cells amount of triethylamine (TEA) at room temperature the unex-
5
(
10 ) with Annexin V-FITC and propidium iodide (PI) was by apop- pected quinazolinone 4 was formed despite the prospective thio-
tosis detection kit [BD Biosciences, San Jose, CA]. The binding of urea derivative 3. In a trial to obtain the open-chain thiourea
Annexin V-FITC and PI was examined using flow cytometry derivative 3, NMR tube reaction was carried out between 1 and 2.
The structure of 3 was confirmed by the presence of O–CH sing-
let signal at dH 3.83; dC 55.76 ppm in the H and C NMR, ester
FACScalibur (BD Biosciences, San Jose, CA). CellQuest software
was used for performing quadrant analysis of co-ordinate
dot plots.
3
1
13
carbonyl at d 167.96 ppm, C¼S at d 179.68 and the two NH sin-
C
C
þ
glets at d 2.87 and 10.31 ppm. The M at 344 m/z confirmed the
H
structure of 3. While in NMR data of 4 the O–CH , as well as one
3
1
3
NH signal disappeared. The ester carbonyl signal in the C NMR
of 3 was replaced by amide carbonyl at d 159.57 ppm in 4 while
2
.3. Molecular docking
C
C¼S appeared at d 176.01 ppm. Methylation of 4 by methyl iod-
C
Docking studies were performed using Molecular Operating
Environment software (MOE, 2015.10) provided by chemical com-
puting group, Canada. The software was used to carry out the
docking of the promising compounds in the receptor’s active site.
The protein crystal structure was obtained from the Protein
ide (MeI) in dry acetone in the presence of anhydrous K CO gave
2
3
the corresponding 2-(methyl thio)quinazolin-4(3H)-one derivative
1
13
5
. Both H and C NMR supported the structure of 5 by the
appearance of S–CH signals at d 2.47; d 15.37 ppm. Moreover,
3
H
C
the C¼S signal at d
C
176.01 ppm in 4 was replaced by C¼N signal
Databank, PDB: 1M17 containing the EGFR enzyme co-crystallized at d 161.09 ppm in 5. On the other hand, the reaction of 1 with
C
with erlotinib. All the water molecules were removed, 3D proton- hydrazine hydrate afforded the reported quinazolinone derivative
2
6
ation was performed. The pocket was determined by the alpha tri- 6 , which was further reacted with MeI to give 7. The structure
angle matcher technique. The Energy Minimization was performed of 6 showed NH proton singlet at d
7.99 and carbon C¼S signal
using MMFF94X force field with RMSD gradient of 0.001 kcal at d
169.27 ppm. However, in 7 the C¼S signal was replaced by
160.94 ppm. The NMR data of 7 also showed the
H
C
ꢁ
1
ꢁ1
mol
Å
and the partial charges were calculated. The co-crystal- C¼N signal at d
C
1
S–CH
3
signals at d
H
2.44; d
C
14.09 ppm. Both H NMR of 6 and 7
H
6.39 and 5.77 ppm, respectively. The
lized ligand was self-docked inside the active site. The compounds
to be docked were drawn on ChemBioOffice 12 and copied as
smiles to MOE followed by docking of 5 and 6.
showed signals for NH
2
at d
Figure 3. Formation of compounds 9 and 10.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
225
MS spectra of 6 and 7 at 193 and 207 m/z, respectively were in
C
d 73.43 ppm indicated a non-substituted OH group. All these
full support of the proposed structures (Scheme 1).
data proved that 11 lack the ring structure present in 9 and 10.
þ
When 1 reacted with 2 in DMF in the presence of a few drops The M at 310 m/z further confirmed the formation of 11.
of TEA yielded the thiazoloquinazolinone 8 rather than the
Treatment of 7 with 2 in EtOH 95%, in the presence of K CO
2 3
expected 4 (Scheme 2, see Supplementary data 1). The structure afforded a mixture of 12 and 13. While when the same reaction
of 8 was confirmed by different spectroscopic data and X-ray crys- was repeated in DMF instead of EtOH the unexpected dimer 14
2
9
tallographic analysis is displayed in Figure 2.
was formed. However, both reactions were expected to give 11
In Scheme 3, the interaction of 3 with hydrazine hydrate in (Scheme 4). The formation of 12 and 13 was assumed to pro-
absolute EtOH gives the expected product 11 in addition to two ceed via addition–elimination mechanisms, as depicted in Figure
other derivatives 9 and 10 that were apparent in TLC. The mech- 4. The NMR data structure of 12 and 13 indicated the disappear-
1
1
anism of formation of 9 and 10 is explained in Figure 3. The H ance of the S–CH signals present in 7. H NMR spectrum of 12
3
13
and C NMR data of 9 and 10 have common features like NH showed NH2 signal at dH 5.50 and OH signal at d 11.62 ppm.
H
þ
doublet at dH 8.01 and 8.12, O¼C–N signal at dC 159.86 and The mass spectrum of 12 showed an M at 177 m/z provided
1
1
61.10 ppm, respectively. Both 9 and 10 showed signal at d
further evidence for replacing the S–CH3 with OH group. In 13
C
54.84 and 152.85 ppm assigned for C¼N carbons attached to signals for an ethoxy group at dH 1.39 (t, J ¼ 7.0, CH ), d
3
C
another hetero atom, respectively. Compound 9 keeps the CH–O 14.61 ppm and d 4.49 (q, J ¼ 7.0, CH ), d 64.79 ppm along with
H
2
C
þ
signals at d
H
6.18 (d, J ¼ 8.0); d
C
81.86 ppm. These signals were
M
at 205 m/z, besides the disappearance of the S–CH signals
3
replaced in 10 by dH 5.86 (d, J ¼ 7.5); d 72.69 ppm assigned for present in 7. The data of 14 indicated the replacement of NH2
C
þ
CH–S. The mass spectrum of 9 showed M at m/z 293 in com- signal by NH at d 3.42 ppm. However, the MS data showed an
H
þ
þ
plete agreement with the proposed structure, while the M at
09 m/z for 10 supported the replacement of one oxygen atom shown in Scheme 4.
with a sulphur atom. The H NMR data of 11 showed signals for
M at 318 m/z, noting that 14 is formed via dimerization of 7, as
3
1
2 3
In Scheme 5, the reaction of 5 with 2 in DMF containing K CO
NH at dH 3.43 (s), NH at d 5.57 (s) and OH at dH 5.73 (s) ppm. afforded the expected product 18 in addition to three other prod-
2
H
Moreover, the chemical shift of the CH–O signals at dH 4.91 (bs); ucts 15, 16 and 17. The mechanism of formation of 15, 16 and
Scheme 4. Synthesis of compounds 12–14.

2
26
M. M. GHORAB ET AL.
1
3
7 is present in Figures 5–7. The NMR data of 15 indicated the elimination of S–CH resulted in 18. The signals of two moieties of
1
13
disappearance of the S–CH3 signals present in 5 and the down- L-norephedrine were overlapped in both H and C NMR spectra.
field shift of the CH–O signals from dH 5.57, d 72.80 ppm in 5 to HR ESI showed a quasi-molecular ion at 430.2127 m/z (calc.
C
þ
d
H
6.18, d
C
82.37 ppm in 15. These data were diagnostic for self- 430.2131) for M þ 1 ion certainly supporting the structure of 18.
cyclization of 5–15 and were further supported by the mass data The NH proton appeared as two broad singlets at 4.15, 4.40 each
þ
that showed M at 278 m/z. The reaction between one molecule integrated for half proton diagnostic for the suggested tautomer-
of 5 and 2 according to Figure 6 resulted in the formation of the isation in the structure. All the assignments of H and C NMR
unique structure of 16. The H and C NMR data of 16 indicated signals were performed based on DEPT 135 as well as 2D NMR
1
13
1
13
the presence of four CH–X groups at d
H
4.73 (d, J ¼ 11.0), d
C
experiments including COSY, HSQC and HMBC (see
3.43; dH 4.79 (d, J ¼ 12.0), d 52.99, dH 5.33 (p, J ¼ 7.0), d 52.64 Supplementary data 2).
5
C
C
and d
H
5.46 (p, J ¼ 7.2), d
C
51.18 ppm. The chemical shift indicated
that none of the heteroatoms is oxygen, the formation of the
complex ring structure involved water elimination and the pres-
3.2. Biological evaluation
ence of two methyl groups at dH 1.23 (d, J ¼ 11.5), d 16.49 and 3.2.1. In vitro cytotoxic activity evaluation
C
þ
d
H
1.24 (d, J ¼ 12.0), d
C
16.74 ppm. The M at m/z 394 was in com- The in vitro cell viability activity of the targeted compounds 4–18
plete agreement with the proposed structure of 16. The NMR data was measured through MTT assay against a panel of cell lines
of 17 indicated the disappearance of the S–CH3 signals and the MDA-MB-231, MCF-7, HepG-2 and HCT-116 human cancer cell
OH signal at dH 5.87 ppm present in 5. The CH–O signals in 17 lines derived from breast, liver and colon tumors. A closer look at
showed a downfield shift to d
H
5.68 (d, J ¼ 8.5), d
C
79.94 ppm Table 1 indicates that compounds 4–18 showed variable IC50 val-
diagnostic for derivatized oxygen atom. Mass spectrum showed ues against the tested cell lines and was compared to erlotinib
þ
M
at 556 m/z consistent with the molecular formula C H N O
4 28 4 4
and staurosporine, as standards. Compound 5 was the most
3
surely prove the dimeric nature of 17. The addition of 2 via the potent against all the cell lines with IC50 ranging from 1.53 to
O
H N
2
NH2
N
+
N
7)
S
OH
(2)
(
OH
OH
O
N
NH2
O
N
NH2
OH
S
N
H
N
O
S
-
H +
-CH S
-
CH S
3
+
-
H
3
O
O
NH2
NH2
OH
N
N
N
O
N
(
12)
(13)
Figure 4. The mechanism of formation of compounds 12 and 13.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
227
Scheme 5. Synthesis of compounds 15–18.
5
.76 mM. Compound 6 takes second place after 5 as a promising selectivity towards MDA-MB-231 followed by HepG-2 cell lines
homologue. Compounds 5, 6 and 15 displayed more potent activ- (Table 2).
ity against MDA-MB-231 cell line in comparison to erlotinib with
IC50 values ¼ 1.53, 1.60 and 2.41 versus 3.73 mM. While the
3.2.2. EGFR kinase assay
remaining compounds showed good to moderate activities
All the newly synthesized compounds, 4–18, were subjected to
towards the tested cell lines. The most potent compounds 5 and
6
were screened against MCF-10A normal breast cell line to deter- EGFR-TK inhibitory assay. Furthermore, a representative compound
mine their selectivity and relative safety towards normal cells. The eliciting superior EGFR inhibition was subjected to cell cycle ana-
compounds showed low cytotoxic effect with IC50¼61.85 and lysis and apoptotic assay to investigate its effect on cell cycle pro-
4
9.21 mM against MCF-10A cell line. Measuring the selectivity gression and apoptosis. Table 1 shows the inhibition data of EGFR
index indicates that compounds 5 and 6 showed the highest (IC50 values) for the examined compounds, erlotinib and
3
0

2
28
M. M. GHORAB ET AL.
Figure 5. Formation of compound 15.
Figure 6. Formation of compound 16.
Figure 7. Formation of compound 17.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
229
Table 1. Antiproliferative and EGFR inhibitory activity of the target compounds 4–18.
a
IC50 (mM)
Cpd no.
Structure
MDA-MB-231
MCF-7
HCT-116
HepG-2
EGFR
4
35.24 ± 0.24
25.55 ± 0.02
23.49 ± 0.05
22.68 ± 0.61
1.39 ± 0.14
5
1.53 ± 0.01
5.43 ± 0.14
5.76 ± 0.11
4.14 ± 0.03
0.76 ± 0.10
6
1.60 ± 0.01
7.23 ± 0.07
10.6 ± 0.19
17.6 ± 0.15
2.13 ± 0.21
7
8
9
23.48 ± 0.06
38.19 ± 0.38
84.15 ± 0.62
68.13 ± 1.21
70.13 ± 0.87
>100
59.25 ± 0.78
49.22 ± 0.49
90.54 ± 0.73
48.29 ± 0.93
47.32 ± 0.47
>100
3.44 ± 0.15
3.74 ± 0.17
9.78 ± 0.31
1
0
21.86 ± 0.04
37.31 ± 0.29
46.27 ± 0.63
35.58 ± 0.36
3.09 ± 0.20
(continued)

2
30
M. M. GHORAB ET AL.
Table 1. Continued.
a
IC50 (mM)
Cpd no.
Structure
MDA-MB-231
MCF-7
HCT-116
HepG-2
EGFR
1
1
16.39 ± 0.06
39.42 ± 0.30
44.15 ± 0.22
33.65 ± 0.26
3.87 ± 0.11
12
13
14
57.78 ± 0.43
81.43 ± 0.27
26.55 ± 0.04
66.31 ± 0.72
50.75 ± 0.29
20.54 ± 0.31
7.39 ± 0.28
8.35 ± 0.32
1.39 ± 0.27
>100
>100
>100
30.12 ± 0.19
29.32 ± 0.16
41.25 ± 0.23
1
5
6
2.41 ± 0.03
19.67 ± 0.13
26.51 ± 0.17
28.46 ± 0.40
76.65 ± 0.81
1.09 ± 0.12
7.83 ± 0.19
1
69.12 ± 0.21
>100
>100
(continued)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
231
Table 1. Continued.
a
IC50 (mM)
Cpd no.
Structure
MDA-MB-231
MCF-7
HCT-116
HepG-2
EGFR
1
7
5.45 ± 0.06
20.14 ± 0.27
18.43 ± 0.20
24.30 ± 0.18
1.02 ± 0.05
1
8
62.94 ± 0.10
62.59 ± 0.18
44.90 ± 0.52
50.0 ± 0.13
0.99 ± 0.04
Erlotinib
3.73 ± 0.01
4.48 ± 0.02
2.78 ± 0.04
3.04 ± 0.20
0.31 ± 0.01
Staurosporine
25.26 ± 0.36
20.32 ± 0.31
10.31 ± 0.28
11.45 ± 0.19
0.82 ± 0.08
a
The results represent the mean of three different experiments ± SE.
Table 3. IC50 of compounds 5 and 6 on cancer cell lines after being subjected
Table 2. The selectivity index of compounds 5 and 6 towards the tested to irradiation.
cell lines.
a
IC50 (mM) after irradiation
Selectivity index (SI)
IC50 (mM)
MCF-10A
Cpd no.
MDA-MB-231
MCF-7
HCT-116
HepG-2
Cpd no.
MDA-MB-231
MCF-7
HCT-116
HepG-2
5
6
a
0.78 ± 0.15
1.04 ± 0.04
2.35 ± 0.18
4.26 ± 0.01
2.28 ± 0.41
6.98 ± 0.25
2.34 ± 0.23
14.26 ± 0.11
5
6
61.85 ± 2.14
49.21 ± 1.52
40.42
30.75
11.39
6.81
10.74
4.64
14.93
23.10
The values represent the mean of three different experiments ± SE.

2
32
M. M. GHORAB ET AL.
Figure 8. Flow cytometry analysis for MDA-MB-231 (A) control cells (B) compound 5.
staurosporine, as reference standards. All analogues showed excel-
lent EGFR inhibition potential ranging from 0.76 to 9.78 mM. The
3
-(1-hydroxy-1-phenylpropan-2-yl)-2-(methylthio)quinazolin-4(3H)-
one 5 demonstrated superior enzyme inhibition better than that
expressed by erlotinib (IC ¼0.76 versus 0.92 mM). Compound 5 is
5
0
the most active compound towards all the tested cell lines and
EGFR inhibitory activity with relative safety towards normal cells
and high selectivity towards MDA-MB-231 cell line. The activity of
5
displayed a remarkable decrease by the replacement of the
methyl mercaptan with the thione group as in 4, while replace-
ment with the 1-hydroxy-1-phenylpropan-2-ylamino group as in
18 demonstrates a narrow range change in activity from 0.76 to
0
.99 mM. Furthermore, replacement of the 1-hydroxy-1-phenylpro-
pan-2-yl in 4 with the amino group as in 6 leads to lowering the
EGFR inhibitory activity (IC50 1.39 versus 2.13 mM). Also,
Figure 9. Histogram showing the effect of compound 5 on cell cycle analysis
using MDA-MB-231 cell line.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
233
replacement of the thione group in 6 with methyl mercaptan 7,
-hydroxy-1-phenylpropan-2-ylamino 11, hydroxy 12 or ethoxy
1
group 13 reduces the activity. Regarding the two homologues 8
and 15, the replacement of sulphur with oxygen greatly enhances
the EGFR activity, while the opposite occurs in 9 and 10. The two
dimers 14 and 17 show very potent activity that demonstrates
that a bulky rigid structure is favourable for binding with
the receptor.
3.2.3. Radiosensitizing activity
Radiotherapy is second to surgery in cancer treatment. The major
drawback of radiotherapy is its inability to differentiate between
cancerous and normal tissues. Radiation causes ionization and
excitation of atoms that result in the generation of short-lived free
radicals. These free radicals can damage proteins and membranes,
3
1,32
leading to single or double DNA strand breaks
. A radiosensitiz-
ing agent can induce tumor sensitization to ionizing radiation,
thus lowering the required dose for treatment. This enhancement
of radiation effects not only control the local tumors but also limit
the metastatic spread. EGFR inhibitors can adopt another mechan-
ism of action by inhibiting accelerated repopulation of tumor cells
during fractionated radiotherapy as they block the membrane
receptors of growth factors or interfere with the signaling path-
3
3,34
ways involved in cell proliferation
.
The ability of the most active compounds 5 and 6 to enhance
gamma radiation-induced tumor cell death was examined. The
results proved the ability of the two compounds to sensitize the
cancerous cells to the lethal effects of ionizing radiation (Table 3).
Compounds 5 and 6 showed enhanced cytotoxicity on all cell
lines after irradiation with a single dose of 8 Gy gamma radiation.
Compound 5 was more potent on all the tested cell lines
with IC50 < 5 mM.
Figure 10. Effect of compound 5 on % of apoptotic cells using Annexin V/
PI assay.
3.2.4. Effect on cell cycle progression
The therapeutic effect of the anticancer agent depends upon its
ability to stop cell cycle progression by arresting cell division at
certain checkpoints promoting apoptosis. These checkpoints exist
3
5,36
at G1-S, S and G2-M phases
. The most potent and selective
compound 5 was chosen to determine its ability to induce apop-
3
7
tosis using MDA-MB-231 cells according to the reported method .
The cells were treated with compound 5 at a concentration equals
to its IC50 value on EGFR (0.76 lM) for 24 h. It is clear from Figures
Figure 11. Induction of apoptosis by compound 5 in MDA-MB-231 cell line.
8
and 9 that compound 5 interfered with the cell cycle in the G2-
Figure 12. The docking pose of erlotinib inside the active site of 1M17.

2
34
M. M. GHORAB ET AL.
Figure 13. 2D & 3D docking poses of compound 5 inside the active site of 1M17.
Figure 14. Superimposition of erlotinib (red) and compound 5 (magenta) showed that they adopt the same orientation inside the active site.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
235
Figure 15. 2D and 3D visuals of compound 6 inside 1M17 active site.
M phase. At that phase, accumulating cells reached 40.39% after 3.2.5. Apoptotic assay
treatment of control MDA-MB-231 cells (6.82%) with compound 5. Phosphatidylserine (PS) exposure on the outer plasma membrane
Furthermore, compound 5 raised the percentage of cells at pri-G1 was detected during apoptosis and forms the basis for Annexin V/
phase by 10 folds to reach 19.23% after being 1.91% in control PI (propidium iodide) double staining assay to detect apoptotic
cells. On the contrary, the cell population in G1 and S phases cell death. At early apoptosis, the cell membrane excludes viability
decrease after treatment with compound 5. So, compound 5 dyes such as PI and permits the determination of apoptotic cell
3
8,39
induces apoptosis through cell cycle arrest in the G2-M phase.
kinetics according to the cell cycle
. To investigate the mode

2
36
M. M. GHORAB ET AL.
Figure 16. Overlaying of compound 6 (blue) and erlotinib (magenta) in the active site of EGFR.
of induced cell death, MDA-MB-231 cells were incubated with 4. Conclusion
compound 5 at 0.76 lM for 24 h. Compound 5 induced apoptosis
In this study, a novel series of quinazolinone and fused quinazoli-
none derivatives synthesized by the aid of L-norephedrine were
obtained. All these compounds showed variable anticancer activity
against MDA-MB-231, MCF-7, HepG-2 and HCT-116 cancer cell
lines and EGFR inhibitory activity comparable to erlotinib. The 3-
(
19.1%) by more than 12 folds over the control (1.54%).
Compound 5 induced early apoptosis by 7.36% and enhanced
late apoptosis by 11.74% compared with the untreated control
cells (Figures 10 and 11).
(1-hydroxy-1-phenylpropan-2-yl)-2-(methylthio)quinazolin-4(3H)-
one 5 and 3-amino-2-thioxo-2,3-dihydroquinazolin-4(1H)-one 6
were the most promising in this series towards the cancer cell
lines and EGFR. Compounds 5 and 6 were further selected to
measure their relative safety and selectivity towards normal cells.
3.3. Molecular docking
Molecular docking of compounds 5 and 6 was performed on the They showed mild cytotoxic activity towards MCF-10A normal cell
line and high selectivity towards MDA-MB-231 cell line. Besides,
they displayed radiosensitizing activity through their ability to sen-
sitize the cancer cells to the lethal effect of gamma irradiation.
The most potent compound in this series, 5, undergoes cell cycle
analysis and annexin V/PI assay to detect apoptotic cell death.
Compound 5 proved to arrest the cell cycle progression at the
G2-M phase, induce early apoptosis and enhance late apoptosis.
Moreover, molecular docking of compounds 5 and 6 showed the
key interactions required for EGFR inhibition. Finally, compounds
active site of EGFR co-crystallized with erlotinib (PDB: 1M17)
4
0
(Figure 12) . The active site of 1M17 consists mainly of these key
amino acids; Met 769, Leu 694, Thr 766, Ala 719, Leu 764, Gln 767,
Leu 768, Pro 770, Phe 771, Gly 772, Leu 820, Thr 830 and Asp 831.
The ligand compounds 5 and 6 were docked into the active site
of the target protein 1M17 and the binding affinities, energy
scores and RMSD values for compounds were recorded. Validation
of molecular docking showed that the RMSD values are within
41
acceptable limits (less than 2 Å) . The best binding affinity with
the lowest energy score for the compounds was computed as
5
and 6 could be considered as promising leads for the develop-
ꢁ
1
ꢁ1
ment of new anticancer and radiosensitizing agents.
ꢁ
10.14 kcal mol
(compound 5) and ꢁ10.03 kcal mol
(com-
pound 6). According to these findings, together with the above-
mentioned biological evaluation, compounds 5 and 6 may act as
effective docking material for EGFR tyrosine kinase. The 2D and
Acknowledgement
3
D visuals of the interaction map for compound 5 can be seen in M. M. Ghorab and A. M. Soliman appreciate the staff members of
Figure 13. The hydrogen bond formation connected the CO of gamma irradiation unit at the National Center for Radiation
Research and Technology (NCRRT) for carrying out the irradiation
process. A. S. Alqahtani is thankful to the Deanship of the
Scientific Research and Research Center, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia.
quinazolinone with Met 769 of the target protein with a length of
.39 Å and Thr 766 by OH with 2.63 Å. Superimposing compound
with erlotinib showed that they adopt the same orientation
inside the active site with RMSD ¼ 1.243 Å (Figure 14). On the
other hand, four conventional hydrogen bond interactions were
observed between compound 6 and the macromolecule 1M17 as
follows; the CO of the quinazolinone with Met 769 and Leu 768
2
5
Disclosure statement
with a recorded distance of 2.54 and 2.79 Å, respectively. In add- No potential conflict of interest was reported by the author(s).
ition to Thr 766 that forms two hydrogen bonds with NH and CS
at a distance of 2.88 and 3.01 Å (Figure 15). Overlaying of erlotinib
and compound 6 can be observed in Figure 16 with RMSD
ORCID
¼
1.236 Å.
Mostafa M. Ghorab
http://orcid.org/0000-0003-4250-0452

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
237
Aiten M. Soliman
http://orcid.org/0000-0001-6562-9828
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