Amr Sonousi1, Rasha A Hassan1, Eman O Osman1, Amr M Abdou2, Soha H Emam1. 1. Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt. 2. Department of Microbiology and Immunology, National Research Centre, Dokki, Giza, Egypt.
Abstract
Nineteen new quinazolin-4(3H)-one derivatives 3a-g and 6a-l were designed and synthesised to inhibit EGFR. The antiproliferative activity of the synthesised compounds was tested in vitro against 60 different human cell lines. The most potent compound 6d displayed superior sub-micromolar antiproliferative activity towards NSC lung cancer cell line NCI-H460 with GI50 = 0.789 µM. It also showed potent cytostatic activity against 40 different cancer cell lines (TGI range: 2.59-9.55 µM). Compound 6d potently inhibited EGFR with IC50 = 0.069 ± 0.004 µM in comparison to erlotinib with IC50 value of 0.045 ± 0.003 µM. Compound 6d showed 16.74-fold increase in total apoptosis and caused cell cycle arrest at G1/S phase in breast cancer HS 578T cell line. Moreover, the most potent derivatives were docked into the EGFR active site to determine their binding mode and confirm their ability to satisfy the pharmacophoric features required for EGFR inhibition.
Nineteen new quinazolin-4(3H)-one derivatives 3a-g and 6a-l were designed and synthesised to inhibit EGFR. The antiproliferative activity of the synthesised compounds was tested in vitro against 60 different human cell lines. The most potent compound 6d displayed superior sub-micromolar antiproliferative activity towards NSC lung cancer cell line NCI-H460 with GI50 = 0.789 µM. It also showed potent cytostatic activity against 40 different cancer cell lines (TGI range: 2.59-9.55 µM). Compound 6d potently inhibited EGFR with IC50 = 0.069 ± 0.004 µM in comparison to erlotinib with IC50 value of 0.045 ± 0.003 µM. Compound 6d showed 16.74-fold increase in total apoptosis and caused cell cycle arrest at G1/S phase in breast cancer HS 578T cell line. Moreover, the most potent derivatives were docked into the EGFR active site to determine their binding mode and confirm their ability to satisfy the pharmacophoric features required for EGFR inhibition.
Cancer is a widespread and lethal noncommunicable disease with rapidly growing incidence and mortality worldwide. Breast cancer is the leading cause of death among women with a reported incidence of approximately 2.3 million and 685,000 deaths worldwide in 2020.Molecular targeting therapy approach, that targets a crucial cancer-related enzyme or receptor, increases the tumour specificity and decreases the side effects. The epidermal growth factor receptor (EGFR) is mainly involved in the growth factor signalling. Abnormal signalling and overexpression of this receptor enhance downstream effects such as cell survival, cell proliferation and angiogenesis which lead to uncontrolled cell proliferation and metastasis, which ultimately promote tumour growth (Figure 1). The EGFR is one of the members of ErbB tyrosine kinase receptors family, and consists of two domains; an extracellular receptor domain connected via a transmembrane region to an intracellular domain with tyrosine kinase function. Inhibition of EGFR by tyrosine kinase inhibitors (TKIs) delays these downstream effects and lead to inhibition of tumour growth. Many breast cancers express 2 × 106 EGFR molecules per cell which is more than 20-fold the expression of EGFR in normal cells,. This overexpression of EGFR in breast cancer as well as other cancer cells including colon and lung cancers made this a potential molecular target for inhibition.
Figure 1.
An illustrative diagram showing the mechanism of action of TKIs as anticancer.
An illustrative diagram showing the mechanism of action of TKIs as anticancer.Three generations of small-molecule EGFR TKIs have been developed so far,. The first-generation, such as gefitinib and erlotinib usually with 4-anilinoquinazoline motifs, achieves initial good responses in the treatment of cancer patients with overexpression of EGFR but unfortunately, resistance to this class was acquired by most patients within 1 year. The second-generation of EGFR TKIs, such as neratinib and afatinib, were developed to overcome the acquired resistance,, however their associated side effects, lead to the design of a novel class of third-generation EGFR TKIs, including osimertinib, rociletinib and olmutinib. This class has been designed and developed to overcome resistance and reduce side effects. The aim of this study is to design, synthesise and evaluate compounds with the same pharmacophoric features as the third generation EGFR TKIs.In previous studies in our lab, we synthesised different TKIs with high therapeutic index. In the present study, the design of the new compounds started by finding common pharmacophoric structural features of the newer generations of EGFR TKI without including the acrylamide moiety. The acrylamide moiety in the second and the third generations gives strong covalent binding to the SH group in Cys797 residue of the receptor. However, these compounds are prone to drug resistance when this irreversible covalent interaction is lost due to C797S mutation,. The common structural features were found to be (A) an upper aryl ring attached to (B) 2-aminopyrimidines or 2-amino arylfusedpyrimidines and the 2-amino is attached to either C) phenoxy group or piperazinyl aryl group (Figure 2). The design of the novel compounds was done to mimic these structural features of the third generation EGFR TKIs to have 2-aminoarylquinazoline linked to an upper aryl ring and the 2-amino is attached to either substituted phenoxy group (3a–d) or piperazinyl aryl (or its isostere) (3e–g). We also designed congener compounds with a 2-thioacetyl spacer between the pyrimidine ring and either phenoxy group (6a–f) or piperazinyl aryl (6g–l) groups to investigate the effect of this spacer on the activity of the compounds (Figure 2). The designed compounds were synthesised and subjected to in vitro screening of their cytotoxic activity against a panel of 60 different cancer cell lines. The compounds were investigated for their ability to inhibit EGFR, their effect on cancer cell cycle and their ability to induce apoptosis. Molecular modelling studies were done as well to rationalise the biological activity of the synthesised compounds.
Figure 2.
The three generations of EGFR inhibitors and the designed target compounds.
The three generations of EGFR inhibitors and the designed target compounds.
Experimental
Chemistry
General
Melting points were obtained on a Griffin apparatus and were uncorrected. Microanalyses for C, H and N were carried out at the Regional Centre for Mycology and Biotechnology, Faculty of Pharmacy, Al-Azhar University. IR spectra were recorded on Shimadzu IR 435 spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of Pharmacy, Cairo University, Cairo, Egypt, and values were represented in cm−1. 1H NMR spectra were carried out on Bruker 400 MHz (Bruker Corp., Billerica, MA, USA) spectrophotometer, Faculty of Pharmacy, Cairo University, Cairo, Egypt. The chemical shifts were recorded in ppm on δ scale, coupling constants (J) were given in Hz and peak multiplicities are designed as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet 13 C NMR spectra were carried out on Bruker 100 MHz spectrophotometer, Faculty of Pharmacy, Cairo University, Cairo, Egypt. Mass spectra were recorded with Advion expression® CMS, Nawah Scientific, Cairo, Egypt. Progress of the reactions were monitored by TLC using precoated aluminium sheet silica gel MERCK 60 F 254 and was visualised by UV lamp. The original NMR spectra of the investigated compounds are provided as supporting information. Compounds 1a,b,, 2a,b, 4a,b, and 5a,b, were prepared as reported.
General procedure for the preparation of 2–(2-(4–(4-substituted) benzylidene) hydrazinyl)-3-phenylquinazolin-4(3H)-one (3a-g)
To a mixture of 2-hydrazino-quinazolinone 2a,b (5 mmol) and the appropriate aryloxybenzaldehydes or dialkylaminobenzaldehydes (5 mmol) in absolute ethanol (50 mL), few drops of acetic acid were added, and the reaction mixture was refluxed for 6 h then cooled. The precipitate, formed upon cooling, was collected by filtration, washed with water crystallised from ethanol to give the corresponding hydrazone derivative 3a-g.
General procedure for the preparation of N'-(4–(4-substituted)benzylidene)-2-((4-oxo-3-aryl-3,4-dihydroquinazolin-2-yl)thio)acetohydrazide derivatives (6a-l)
To a mixture of acetohydrazide derivatives 5a,b (5 mmol) and the appropriate aryloxybenzaldehydes or dialkylaminobenzaldehydes (5 mmol) in ethanol (50 mL), few drops of acetic acid were added, and the reaction mixture was refluxed for 6 h then cooled. The precipitate was filtered, washed with water crystallised from ethanol to give the corresponding acetohydrazide derivatives 6a-l.
The biological assays were carried out according to the previously reported procedures and have been provided in the Supplementary Materials; antiproliferative activity screening and five dose concentrations assay by NCI, in vitro EGFR inhibitory assay, cell cycle analysis, apoptosis assay, caspase-3 enzyme assay.
Molecular modelling studies
The crystallographic structure of EGFR protein (PDB: 1M17) was obtained from the protein data bank website, (http://www.pdb.org) with resolution of 2.60 Å. All the molecular modelling studies were carried out using Molecular Operating Environment (MOE 2020.09; Chemical Computing Group, Canada) as the computational software. The hydrogen atoms were added, the protonation states of the amino acid residues were assigned, and the partial charges of atoms were added using Protonate 3D algorithm. Compounds were modelled using MOE builder, and the structure was energy minimised using the MMFF94x force field. Using the MOE induced-fit Dock tool, docking studies of the synthesised compound into the active site was done and the final docked complexes of ligand–enzyme was selected according to the criteria of binding energy score combined with geometrical matching quality.
Statistical analysis
Data are represented as mean ± SD. Significant differences between groups were analysed by using Graphpad Prism 9.1.0. Differences were considered significant at p < 0.05.
Result and discussion
Nineteen new quinazolin-4(3H)-one derivatives 3a–g and 6a–l were synthesised according to Scheme 1. The key starting compounds 3-phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (1a) and 3–(4-chlorophenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (1b) were obtained in good yields by reacting anthranilic acid with the appropriate aryl isothiocyanate in refluxing ethanol in presence of triethylamine as catalyst,. Compounds 1a,b were then treated with hydrazine hydrate 99% as reported to yield 2-hydrazinyl-3-phenylquinazolin-4(3H)-one (2a) and 3–(4-chlorophenyl)-2-hydrazinylquinazolin-4(3H)-one (2b). Furthermore, heating the obtained hydrazine derivatives 2a,b with 4-substituted benzaldehyde derivatives in ethanol as solvent in the presence of few drops of glacial acetic acid afforded the novel target compounds 3a–g. The 1H NMR spectra of the compounds 3a–g showed D2O exchangeable singlet signal due to NH at the range δ 10.49–10.61 ppm. Moreover, singlet signal at the range δ 7.93–8.09 ppm was present due to the characteristic azomethine protons.
Scheme 1.
Synthesis of quinazolin-4(3H)-one derivatives 3a–g and 6a–l. Reagents and conditions: (i) NH2NH2.H2O, ethanol, reflux, 12 h; (ii) Ethyl bromooacetate, dry acetone, anhydrous K2CO3, RT, 8 h; (iii) NH2NH2.H2O, ethanol, reflux, 6 h; (iv) 4-Substituted benzaldehyde, ethanol, drops of glacial acetic acid, reflux, 6 h.
Synthesis of quinazolin-4(3H)-one derivatives 3a–g and 6a–l. Reagents and conditions: (i) NH2NH2.H2O, ethanol, reflux, 12 h; (ii) Ethyl bromooacetate, dry acetone, anhydrous K2CO3, RT, 8 h; (iii) NH2NH2.H2O, ethanol, reflux, 6 h; (iv) 4-Substituted benzaldehyde, ethanol, drops of glacial acetic acid, reflux, 6 h.On the other hand, alkylation of 2-thioxo-2,3-dihydroquinazolin-4(1H)-one derivatives 1a,b with ethyl bromoacetate at room temperature in dry acetone containing anhydrous potassium carbonate afforded ethyl 2-((4-oxo-3-aryl-3,4-dihydroquinazolin-2-yl)thio)acetate 4a,b,. The ester derivatives 4a,b were further reacted with hydrazine hydrate 99% to afford the corresponding acetohydrazide derivatives 2-((4-oxo-3-aryl-3,4-dihydroquinazolin-2-yl)thio)acetohydrazide 5a,b,. Compounds 5a,b were finally reacted with 4-substituted benzaldehyde derivatives to yield the desired hydrazone derivatives 6a–l. The 1H NMR spectra of derivatives 6a–l revealed that the hydrazones existed in a keto amide/enol amide mixture (Figure 3). 1H NMR spectra of series 6a–l showed two singlet signals with an integration of 2H due to SCHC=O protons at the ranges of δ 4.45–4.47 ppm and δ 3.97–4.01 ppm corresponding to keto amide and enol amide tautomers, respectively. Moreover, two singlet signals with an integration of 1H in 1H NMR spectra of 6a–l appeared due to the azomethine protons of both tautomers at the ranges of δ 8.07–8.24 and δ 7.90–8.04 ppm. Additionally, two D2O exchangeable peaks due to OH proton in enol and NH in keto tautomers with 1H total integration appeared at the ranges of δ 11.49–11.73 ppm and δ 11.39–11.64 ppm, respectively. On the other hand, the appearance of the OCH3 group in derivatives 6c and 6f as two singlet signals with total integration of 3H at δ 3.77–3.78 ppm and δ 3.76–3.77 ppm is in concordance with the presence of tautomerism. 13 C NMR spectra of derivatives 6a–l showed two peaks at the ranges δ 35.8–35.9 ppm and δ 34.6–34.9 ppm corresponding to SH2C=O in enol and keto tautomers, respectively.
Figure 3.
Tautomerism in hydrazone derivatives 6a–f and 6g–l.
Tautomerism in hydrazone derivatives 6a–f and 6g–l.
Evaluation of antiproliferative activity against a panel of 60 human cancer cell lines
Two series of substituted quinazolinone derivatives 3a–g and 6a–l were evaluated for their antiproliferative activity at a single dose (10 μM) using 60 human cancer cell lines, by the National Cancer Institute (NCI), USA. The screening was achieved under the Developmental Therapeutic Program (DTP). NCI cell lines include leukaemia, melanoma, and cancers of the breast, kidney, ovarian, colon, central nervous system (CNS), prostate, and non-small cell (NSC) lung. The growth inhibition percentage (GI%) representing the in vitro antiproliferative activity was illustrated in Table 1.
Table 1.
In vitro growth inhibition % (GI%) of the synthesised compounds 6a–6l against a panel of 60 tumour cell lines at 10 μM.
Subpanel
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
Leukaemia
CCRF-CEM
49.57
43.1
–
> 100
–
36.46
–
–
–
–
–
–
HL-60(TB)
–
–
–
–
–
–
–
–
–
–
–
–
K-562
38.79
37.52
–
77.02
–
41.57
26.76
–
–
26.42
–
–
MOLT-4
41.34
44.47
–
> 100
–
38.29
28.47
–
–
–
–
–
RPMI-8226
26.75
31.73
–
75.28
–
14.91
17.56
–
–
–
–
–
SR
29.66
26.64
–
67.08
–
27.19
24.73
–
–
25.23
–
–
NSC lung cancer
A549/ATCC
58.27
57.81
–
83.53
–
52.97
15.14
–
–
50.65
–
–
EKVX
40.95
39.26
–
59.16
–
30.59
20.43
–
–
17.94
–
–
HOP-62
40.92
36.11
–
67.44
–
26.68
–
–
–
81.63
–
–
HOP-92
33.3
26.96
–
97.41
–
22.66
19.49
18.69
–
37.67
–
–
NCI-H226
27.88
22.43
–
24.44
–
20.29
–
–
–
61.77
–
–
NCI-H322M
15.51
–
–
–
–
15.35
–
–
–
23.03
–
–
NCI-H460
67.86
71.98
–
> 100
–
88.8
–
–
–
90.26
–
–
NCI-H522
50.38
51.53
–
79.34
13.4
48.7
15.3
–
–
–
–
–
Colon cancer
COLO-205
50.76
55.61
–
85.4
–
27.18
–
–
–
17.87
–
–
HCC-2998
–
–
–
79.7
–
16.65
–
–
–
–
–
–
HCT-116
94.38
92.19
–
> 100
–
86.66
15.51
–
–
63.1
–
–
HCT-15
39.16
36.28
–
89.62
–
43.95
16.21
–
–
32.2
–
–
HT29
40.85
61.82
–
98.24
–
83.93
–
–
–
39.1
–
–
KM12
16.31
23.73
–
82.6
–
22.83
–
–
–
3.59
–
–
SW-620
56.93
70.37
–
97.38
–
55.6
–
–
–
34.12
–
–
CNS cancer
SF-268
–
15.72
–
44.24
–
42.06
–
–
–
65.26
–
–
SF-295
16.22
16.28
–
35.28
–
32.32
–
–
–
23.56
–
–
SF-539
31.48
31.3
–
30.33
–
67.83
–
–
–
58.67
–
–
SNB-19
66.78
60.15
–
71.51
–
38.45
–
–
–
56.75
–
–
SNB-75
39.91
42.17
–
42.06
–
58.37
19.86
–
–
20.14
–
–
U251
84.83
78.53
–
> 100
–
63.53
–
–
–
49.41
–
–
Melanoma
LOX IMIV
86.02
79.01
–
> 100
–
85.33
–
–
–
56.49
–
15.15
MALME-3M
48.74
45.07
–
> 100
–
36.13
–
–
–
35.79
–
–
M14
51.16
43.73
NT
> 100
NT
NT
–
NT
NT
NT
NT
NT
MDA-MB-435
15.38
19.23
–
74.95
–
–
–
–
–
–
–
–
SK-MEL-2
13.13
–
–
45.68
–
–
–
–
–
–
–
–
SK-MEL-28
37.58
29.11
–
96.83
–
–
–
–
–
15.16
–
–
SK-MEL-5
28.84
40.93
–
89.11
–
21.98
–
–
–
–
–
–
UACC-257
–
–
–
41.0
–
–
–
–
–
–
–
–
UACC-62
33.42
31.24
–
99.57
–
38.11
–
–
–
26.53
–
–
Ovarian cancer
IGROV1
50.89
34.45
–
79.42
–
51.35
–
–
–
44.51
–
–
OVCAR-3
–
–
–
–
–
> 100
–
–
–
67.19
–
–
OVCAR-4
26.25
18.26
–
98.3
–
27.45
–
–
–
41.44
–
–
OVCAR-5
18.55
–
–
29.36
–
18.76
–
–
–
23.12
–
–
OVCAR-8
40.84
36.67
–
86.57
–
–
–
–
–
34.7
–
–
NCI/ADR-RES
47.27
44.83
–
84.26
–
48.89
–
–
–
60.15
–
–
SK-OV-3
16.8
15.49
–
66.17
–
33.7
–
–
–
69.91
–
–
Renal cancer
786-0
46.79
54.01
–
> 100
–
74.84
–
–
–
92.8
–
–
A498
34.29
17.71
17.18
> 100
15.7
27.11
–
15.82
10.22
21.52
–
32.61
ACHN
25.52
–
–
36.52
–
92.17
–
–
–
82.62
–
–
CAKI-1
40.81
29.24
NT
66.99
NT
NT
21.97
NT
NT
NT
NT
NT
RXF 393
57.96
37.84
NT
> 100
NT
NT
–
NT
NT
NT
NT
NT
SN12C
73.08
59.26
–
54.75
–
47.29
–
–
–
37.62
–
–
TK-10
–
–
–
60.33
–
–
–
–
–
–
–
–
UO-31
18.82
–
–
–
20.82
52.26
23.31
–
–
46.97
–
–
Prostate cancer
PC-3
41.33
28.25
–
99.2
–
36.32
18.52
–
–
21.44
–
–
DU-145
61.56
61.67
–
86.91
–
50.84
–
–
–
70.89
–
–
Breast cancer
MCF7
35.46
31.12
–
84.47
–
50.72
16.22
–
–
32.51
–
–
MDA-MB-231/ATCC
50.77
49.15
–
69.61
–
53.27
—
–
–
28.05
–
–
HS 578T
41.12
39.2
–
78.62
–
> 100
–
–
–
62.12
–
–
BT-549
30.09
26.93
–
> 100
–
–
–
–
–
–
–
–
T-47D
17.5
17.61
–
69.86
–
62.15
–
–
–
25.43
–
–
MDA-MB-468
28.41
34.89
–
> 100
–
33.16
–
–
–
20.54
–
–
Mean
37.29
34.78
< 0
80.23
< 0
42.46
4.28
< 0
< 0
34.19
< 0
< 0
–: growth inhibition below 15%.; NT: not tested.
Bold numbers: growth inhibition above 70%.
In vitro growth inhibition % (GI%) of the synthesised compounds 6a–6l against a panel of 60 tumour cell lines at 10 μM.–: growth inhibition below 15%.; NT: not tested.Bold numbers: growth inhibition above 70%.The series 3a–g, representing 2-(substitutedbenzylidene)hydrazinyl quinazolin-4(3H)-ones showed low to no activity against most of the investigated cancer cell lines (Supplementary data). In contrast, five derivatives of the acetohydrazide series 6a–l displayed potent broad-spectrum antiproliferative activity against most of the examined cancer cell lines with mean growth percentage of range 34.19 − 80.23%.4-chlorophenoxybenzylidene acetohydrazide derivative 6a exhibited broad-spectrum antiproliferative activity with mean GI% of 37.29. It showed significantly potent antiproliferative activity against colon cancer HCT-116 (GI% 94.38); CNS cancer U251 (GI% 84.83); melanoma LOX IMIV (GI% 86.02) and renal cancer SN12C (GI% 73.08). It had also demonstrated excellent-to-moderate antiproliferative activity against 33 cancer cell lines with GI% in the range of 67.86% to 30.09%. Moreover, compound 6b with 4-bromophenoxybenzylidene acetohydrazide demonstrated broad-spectrum antiproliferative activity with mean growth inhibition of 34.78%. It showed significantly potent antiproliferative activity against NSC lung cancer NCI-H460; colon cancer (HCT-116 and SW-620); CNS cancer U251 and melanoma LOX IMIV with GI% 71.98%, 92.19%, 70.37%, 78.53% and 79.01%, respectively. It revealed excellent-to-moderate antiproliferative activity against 29 cancer cell lines with growth inhibition in the range of 61.82–31.2%.Compound 6c with 4-methoxyphenoxy substitution showed no activity against all tested cancer cell lines. Compound 6d with (4-chlorophenoxy)benzylidene)-3–(4-chlorophenyl)quinazolinone demonstrated significantly potent to excellent broad-spectrum antiproliferative activity with mean GI% 80.23. It was accordingly evaluated at five dose concentrations by NCI to define its dose-response behaviour and determine its GI50, TGI, and LC50 values. (Values were represented in Table 2). Compound 6d showed significantly potent antiproliferative activity against 24 cancer cell lines with GI% ranging between 99.57% and 71.51%. Also, compound 6d showed significantly potent antiproliferative activity with inhibition percentage above 90% with NSC lung cancer HOP-92 (GI% 97.41); colon cancer (HT29 and SW-620 with GI% 98.24 and 97.38, respectively); melanoma (SK-MEL-28 and UACC-62 with GI% 96.83 and 99.57 respectively); ovarian cancer OVCAR-4 (GI% 98.3) and prostate cancer PC-3 (GI% 99.2). Additionally, compound 6d displayed lethal activity against 13 of the tested cancer cell lines with growth inhibition above 100%.
Table 2.
The values of GI50, TGI and LC50 of compound 6d at 5 doses against 60 cell line panel.
6d
Compound
GI50
TGI
LC50
Leukaemia
CCRF-CEM
3.09
48.5
>100
HL-60(TB)
16.0
37.0
85.7
K-562
4.15
20.2
>100
MOLT-4
2.29
9.23
44.7
RPMI-8226
2.98
15.8
>100
SR
1.84
6.86
31.9
NSC lung cancer
A549/ATCC
3.25
14.0
83.3
EKVX
3.1
13.9
63.6
HOP-62
1.69
4.05
9.69
HOP-92
2.07
5.62
22.8
NCI-H226
2.46
6.59
33.2
NCI-H322M
3.02
15.0
90.8
NCI-H460
0.789
2.6
7.78
NCI-H522
2.34
8.31
42.6
Colon cancer
COLO 205
2.79
8.74
91.6
HCC-2998
1.92
3.81
7.57
HCT-116
1.55
3.74
9.03
HCT-15
2.41
7.48
28.5
HT29
2.14
11.1
>100
KM12
2.04
5.0
26.6
SW-620
1.81
7.84
42.4
CNS cancer
SF-268
3.85
23.9
>100
SF-295
3.61
13.3
36.4
SF-539
2.54
6.32
21.5
SNB-19
3.36
12.7
53.2
SNB-75
3.99
29.2
>100
U251
1.65
3.69
8.23
Melanoma
LOX IMVI
1.29
2.59
5.2
MALME-3M
1.38
2.77
5.55
M14
1.58
3.47
7.58
MDA-MB-435
1.93
4.56
13.0
SK-MEL-2
2.86
8.93
41.3
SK-MEL-28
1.51
3.01
6.01
SK-MEL-5
1.75
3.51
7.07
UACC-257
4.62
22.5
>100
UACC-62
1.59
3.18
6.38
Ovarian cancer
IGROV1
1.51
3.28
7.12
OVCAR-3
2.75
9.55
38.9
OVCAR-4
1.89
4.57
17.5
OVCAR-5
2.27
6.44
32.2
OVCAR-8
2.41
6.56
>100
NCI/ADR-RES
1.64
3.56
7.76
SK-OV-3
3.26
11.9
49.4
Renal cancer
786-0
2.51
6.41
26.5
A498
1.94
4.34
9.74
ACHN
4.36
15.2
39.0
CAKI-1
3.06
12.0
35.4
RXF 393
1.7
3.14
5.81
SN12C
1.74
4.14
9.84
TK-10
5.97
40.2
>100
UO-31
7.47
21.1
47.1
Prostate cancer
PC-3
2.58
9.32
>100
DU-145
3.52
20.4
>100
Breast cancer
MCF7
2.01
5.5
25.6
MDA-MB-231/ATCC
1.79
3.9
8.51
HS 578T
2.17
6.36
>100
BT-549
3.28
12.2
36.2
T-47D
2.09
5.76
49.5
MDA-MB-468
2.08
5.08
18.8
The values of GI50, TGI and LC50 of compound 6d at 5 doses against 60 cell line panel.Replacing 4-chlorophenoxy with 4-bromophenoxy in derivative 6e resulted in abolishing the antiproliferative activity. On the other hand, derivative 6f with 4-methoxyphenoxy substitution displayed antiproliferative activity (mean GI% of 42.46). Compound 6f exhibited significantly potent antiproliferative activity against 6 cancer cell lines representing NSC lung cancer NCI-H460; colon cancer (HCT-116 and HT29); melanoma LOX IMIV and renal cancer (786–0 and ACHN) with GI% 88.8%, 86.66%, 83.93%, 85.33%, 74.84% and 92.17%, respectively. It also showed moderate activity against 27 cell lines with growth inhibition percentage ranging from 67.83 to 30.59%. In addition, compound 6f showed lethal activity against ovarian cancer OVCAR-3 and breast cancer HS 578 T with growth inhibition above 100%.Derivative 6j with N'-(4-(piperidin-1-yl)benzylidene)-3–(4-chlorophenyl)-quinazolinone acetohydrazide showed antiproliferative activity with mean GI% of 34.19. It showed significantly potent antiproliferative activity against 5 cell lines including NSC lung cancer (HOP-62 and NCI-H460); renal cancer (786–0 and ACHN) and prostate DU-145 with GI% 81.63%, 90.26%, 92.8%, 82.62% and 70.89% respectively. It showed moderate antiproliferative activity against 23 cell lines with GI% range of 69.91–32.2%. Finally, derivatives 6 h, 6i, 6k and 6 l did not show activity against all cell lines.The most remarkable effects to be concluded in the structure variations of the synthesised derivatives and their antiproliferative activity are represented in Figure 4. The incorporation of thioacetyl linker at position 2 of the quinazolinone ring was found to be essential for the antiproliferative activity as most of the synthesised compounds with thioacetyl bridge in series 6a–l were found to have of high anticancer activity while compounds 3a–g, lacking the thioacetyl bridge, showed no promising results. This may be due to the increased flexibility introduced by thioacetyl spacer, which enables the compounds to fit better into the receptor.
Figure 4.
Structure activity relationship for synthesised quinazolinone derivatives correlated with their cytotoxic activity.
Structure activity relationship for synthesised quinazolinone derivatives correlated with their cytotoxic activity.In series 6a–l, general better antiproliferative activity were displayed by compounds bearing chloro atom at the para-position of the quinazolinone 3-phenyl ring as demonstrated by the relatively high GI percentage values of compounds 6d (mean GI% = 80.23%), 6f (mean GI% = 42.46%) and 6j (mean GI% = 34.19%) over their unsubstituted analogues 6a (mean GI% = 37.29%), 6c (mean GI% = 0%) and 6 g (mean GI% = 4.28%), respectively. This observation was in accordance with the previously reported study in other related compounds. It was also observed that the substituted phenoxy derivatives 6a–f have generally better GI% values than the corresponding piperazinyl (or its isostere) aryl group 6g–l.
Evaluation of in vitro antiproliferative activity of compound 6d at 5 dose concentrations
Compound 6d was found to be the most effective anticancer agent in this study based on preliminary single dose (10 μM) screening results. It demonstrated promising efficacy against a variety of cancer cell lines, with mean growth inhibition of 80.23% (Table 1). Accordingly, compound 6d was further subjected to five dose concentrations assay (0.01, 0.1, 1, 10 and 100 μM) to detect its dose-response behaviour and calculate the values of GI50 (the dose that inhibit 50% of cell growth in comparison to control), TGI (the dose that completely inhibit growth), and LC50 (the dose that kill 50% of the cells). The values are presented in Table 2. The obtained results showed that, compound 6d displayed superior sub-micromolar activity towards NSC lung cancer cell line NCI-H460 with GI50= 0.789 µM, however it showed high lethality with this cell line (LC50= 7.78 µM). Compound 6d exhibited also potent and broad-spectrum antiproliferative activity against most of the tested cancer cell lines, with GI50 values in the range of 1.29–5.97 µM, in addition to moderate antiproliferative activity against leukaemia cell line HL-60(TB) with GI50= 16.0 µM. Compound 6d also exhibited high cytostatic activity (TGI range: 2.59–9.55 µM) against 40 cancer cell lines. Moreover, 6d demonstrated good to moderate cytostatic activity against the rest of cell lines with TGI range of 11.1–48.5 µM. Compound 6d exhibited remarkable differences between its cytotoxic indicator (LC50) and cytostatic markers (GI50 and TGI) against colon cancer HT29; ovarian cancer OVCAR-8; prostate cancer PC-3 and breast cancer HS 578T, which indicates a wide therapeutic index.
In vitro EGFR kinase inhibitory assay
The most potent compounds 6a, 6b, 6d, 6f and 6j, with promising antiproliferative activity with mean inhibition range of 34.19–80.23%, were assayed for their EGFR inhibition. The results were summarised in Table 3 and Figure 5 as 50% inhibition concentration value (IC50) calculated from the concentration inhibition response curve. In this assay, erlotinib is used as positive control. Compound 6d potently inhibited EGFR with IC50 = 0.069 ± 0.004 µM in comparison to erlotinib with IC50 value of 0.045 ± 0.003 µM. It was obvious that there was non-significant difference between IC50 values scored by both compound 6d and erlotinib. Compound 6f with IC50 = 0.133 ± 0.008 µM, showed nearly half the potency of EGFR inhibition compared to compound 6d. Moreover, compound 6b was nearly equipotent in EGFR inhibition with compound 6j with IC50 values of 0.487 ± 0.030 and 0.478 ± 0.029 µM, respectively. Compound 6d, showed nearly three times the potency of EGFR inhibition compared to compound 6a with IC50 = 0.202 ± 0.012 µM. The antiproliferative activity of the compounds correlated with their high ability to inhibit EGFR. These biological results indicate that compound 6d is a promising antiproliferative agent with EGFR inhibitory activity.
Table 3.
EGFR kinase inhibitory activity of compounds 6a, 6b, 6d, 6f and 6j compared with erlotinib
Compound
EGFR IC50 (µM ± SD)a
6a
0.202 ± 0.012
6b
0.487 ± 0.030
6d
0.069 ± 0.004
6f
0.133 ± 0.008
6j
0.478 ± 0.029
Erlotinib
0.045 ± 0.003
aIC50 are presented as mean of three independent experiments.
Figure 5.
Graphical illustration for IC50 inhibition of EGFR of compounds 6a, 6b, 6d, 6f and 6j compared to erlotinib. Statistical significance was analysed by one-way ANOVA and Tukey’s multiple comparisons test (ns: non-significant, ***p < 0.001, ****p < 0.0001).
Graphical illustration for IC50 inhibition of EGFR of compounds 6a, 6b, 6d, 6f and 6j compared to erlotinib. Statistical significance was analysed by one-way ANOVA and Tukey’s multiple comparisons test (ns: non-significant, ***p < 0.001, ****p < 0.0001).EGFR kinase inhibitory activity of compounds 6a, 6b, 6d, 6f and 6j compared with erlotinibaIC50 are presented as mean of three independent experiments.
Cell cycle analysis
Most cytotoxic drugs exert their antiproliferative activity by disturbing some checkpoints in the cell cycle. These checkpoints are distinct stages in the cell cycle whose disruption causes growth termination. The cell cycle distribution was measured by DNA flow cytometric analysis, upon incubation of HS578T cells treated with compound 6d for 24 h at its IC50 concentration (2.17 µM). The effect of compound 6d on the cell population in different cell phases was recorded and presented in Figure 6. The results were compared to the cell cycle analysis of breast cancer HS 578 T cells as untreated control. The proportions of cells in both the G0-G1 phase and the S phase were significantly increased by 1.12-fold. Moreover, significant decrease in the cell population occurred at the G2/M phase with 52%. In comparison to control, the cell population in the pre-G1 phase significantly increased by 16.74-fold. These results showed that, compound 6d caused induction of cell cycle arrest in breast cancer HS 578 T cells at the G1/S phase.
Figure 6.
Cell cycle analysis in breast HS 578T cell line. (A) Control HS 578T; (B) Treatment with 6d; (C) Graphical diagram for distribution of cell cycle analysis in treated and untreated control cells. Statistical significance was analysed by two-way ANOVA and Tukey’s multiple comparisons test (**p < 0.01, ****p < 0.0001).
Cell cycle analysis in breast HS 578T cell line. (A) Control HS 578T; (B) Treatment with 6d; (C) Graphical diagram for distribution of cell cycle analysis in treated and untreated control cells. Statistical significance was analysed by two-way ANOVA and Tukey’s multiple comparisons test (**p < 0.01, ****p < 0.0001).
Apoptosis assay
Annexin-V flow cytometry assay is an effective technique to determine apoptosis and for discrimination of programmed apoptosis and non-specific necrosis,. Annexin V is a protein with high affinity for phosphatidylserine (PS), a cell membrane component that translocate from the plasma membrane to the surface of the cell during apoptosis. Annexin-V and propidium iodide (PI) dual staining allows for the differentiation of live cells, early/late apoptotic cells, and necrotic cells. Fluorescent Annexin V conjugate can be used to identify PS on the cell surface. PI only enters dead cells and stains DNA. The effect of compound 6d on cell apoptosis was examined in breast cancer HS 578T cell line using a dual staining assay. The percentage of apoptotic cells increased significantly in early (from 0.37% to 19.24%) and late (from 0.22% to 13.45%) apoptotic phases (Table 4 and Figure 7). Compound 6d increased total apoptosis significantly by 16.74-fold compared to the control.
Table 4.
Annexin V/PI dual staining assay in breast HS 578T cells; Distribution of apoptotic cells after treatment with compound 6d.
Comp.
Apoptosis
Necrosis
Total
Early
Late
6d
39.02
19.24
13.45
6.33
Control
2.33
0.37
0.22
1.74
Figure 7.
Effect of compound 6d on Annexin V positive staining percentage in breast cancer HS 578T cells. (A) Control HS 578T; (B) Cells treated with 6d; (C) Graphical diagram for percentage of apoptotic and necrotic cells in treated cells and untreated control cells. Statistical significance was analysed by two-way ANOVA and Tukey’s multiple comparisons test (***p < 0.001, ****p < 0.0001).
Effect of compound 6d on Annexin V positive staining percentage in breast cancer HS 578T cells. (A) Control HS 578T; (B) Cells treated with 6d; (C) Graphical diagram for percentage of apoptotic and necrotic cells in treated cells and untreated control cells. Statistical significance was analysed by two-way ANOVA and Tukey’s multiple comparisons test (***p < 0.001, ****p < 0.0001).Annexin V/PI dual staining assay in breast HS 578T cells; Distribution of apoptotic cells after treatment with compound 6d.
Evaluation of caspase-3 activation
Apoptosis is triggered by the activation of caspases, particularly caspase-3, which is an initiator caspase responsible for apoptosis. Therefore, the induction of apoptosis by compound 6d was examined by the evaluation of caspase-3 activation in breast HS 578T cancer cell line. The effect of erlotinib as well as untreated cell line was used as positive and negative control, respectively. Compound 6d and erlotinib significantly increased caspase-3 level by 10.75- and 8.78-fold, respectively when compared to negative control Figure 8. These results revealed that compound 6d might induce apoptosis via a caspase-dependent mechanism.
Figure 8.
Caspase-3 activity in breast HS 578T cells: Effect of compound 6d compared to erlotinib. Statistical significance was analysed by one-way ANOVA (***p < 0.001, ****p < 0.0001).
Caspase-3 activity in breast HS 578T cells: Effect of compound 6d compared to erlotinib. Statistical significance was analysed by one-way ANOVA (***p < 0.001, ****p < 0.0001).
Molecular docking study
Docking study was carried out for the most potent synthesised compounds into the EGFR protein complexed with erlotinib (PDB: 1M17) using MOE (2022.09) software. The aim of this study was to investigate the interactions between the EGFR-TK and the highly active derivatives, 6a, 6b, 6d, 6f and 6j. The study began by redocking the co‐crystallised ligand, erlotinib, starting from a two‐dimensional structure and using the same protocol for preparation and analysis. The most active derivatives were then docked, and the resulted scores and the binding interactions are included along with the Lipinski parameters in Table 5. Erlotinib fits into the gorge of the active site of EGFR-TK and binds to Cys751 via a water bridge, the N1 atom of the erlotinib accepts a H-bond from the Met769 amide nitrogen. Erlotinib C-2 hydrogen is in close proximity and interacts with Gln767. The anilino ring is coplanar with the quinazoline ring and shows arene-cation interaction with Lys721 (Figure 9). Compounds 6a, 6b, 6d, 6f and 6j parameters were aligned with the Lipinski parameters, except for the logP parameter. Generally, compounds 6a, 6b, 6d, 6f and 6j were able to overlay as erlotinib with the quinazolinone ring inhabiting the same space of the quinazoline ring of erlotinib. The docking simulation showed that the least active compound 6b is less binding to the active site with only 2 interactions: [HB (Cys751) and Arene-H (Val702)], while erlotinib has 4 interactions: [HB (Cys751), HB (Gln767), HB (Met769) and Arene-cation (Lys721)]. On the other hand, the docking of the most active compound 6d (Figure 10) showed a comparable binding profile with the active site as the co-crystalized drug (erlotinib). Compound 6d forms hydrogen bond with Cys751 via the water bridge and H-π interactions with Val702 which help stabilising the quinazolinone ring conformation. The p-chloro phenyl was able to overlay with the anilino ring of erlotinib and also interacts by H-π stacking with Lys721. The chlorine atom forms halogen bond with the carboxylate group of Glu738 which might be the rationale behind the higher in vitro EGFR enzymatic inhibition of 6d when compared to its dechlorinated congener 6a. (Docking poses of compounds 6a, 6b, 6f and 6j are included in supplementary data)
Table 5.
The docking score and Lipinski parameters of compounds 6a, 6b, 6d, 6f and 6j.
Compound number
Docking score (PDB: 1M17) (kcal/mol)
HBA(≤ 10)
HBD(≤ 5)
LogP(−4.0–5)
PSA(0–150) Å2
Interactions
6a
−8.27
5
1
6.66
83.4
HB (Cys751)Arene-H (Val702)
6b
−8.37
5
1
6.77
83.4
HB (Cys751)Arene-H (Val702)
6d
−8.07
5
1
7.32
83.4
HB (Cys751)Arene-H (Val702)Arene-H (Lys721)Halogen bond (Glu738)
6f
−7.55
6
1
6.67
92.6
HB (Lys721)Arene-H (Val702)Arene-H (Leu694)Halogen bond (Glu738)
6j
−7.98
5
1
5.86
77.4
HB (Cys751)Arene-H (Val702)Arene-H (Lys721)Halogen bond (Glu738)
(A) 2D diagram of erlotinib interactions with EGFR binding pocket; (B) 3D overlay of the co-crystallised erlotinib (black) inhibitor and its re-docked pose (magenta) in the binding site of EGFR-TK showing minimum deviation of the docked pose from the co-crystallised one.
Figure 10.
(A) 2D diagram and (B) 3D representation of molecular docking of compound 6d (magenta) in the binding site of EGFR (PDB: 1M17).
(A) 2D diagram of erlotinib interactions with EGFR binding pocket; (B) 3D overlay of the co-crystallised erlotinib (black) inhibitor and its re-docked pose (magenta) in the binding site of EGFR-TK showing minimum deviation of the docked pose from the co-crystallised one.(A) 2D diagram and (B) 3D representation of molecular docking of compound 6d (magenta) in the binding site of EGFR (PDB: 1M17).The docking score and Lipinski parameters of compounds 6a, 6b, 6d, 6f and 6j.
Conclusion
Nineteen quinazolin-4-one derivatives were synthesised to mimic the third generation EGFR TKIs. The antiproliferative activity were assessed for all the prepared compounds. The results showed the significance of the thioacetyl linker for the antiproliferative activity. The compounds showed promising results with 6d exhibiting high inhibition of EGFR with IC50 = 0.069 ± 0.004 µM which is comparable with the positive control. In addition, 6d demonstrated an excellent in vitro antiproliferative activity with mean growth inhibition of 80.23% and was chosen by the National Cancer Institute (NCI), Maryland, USA for further investigation in which compound 6d exhibited potent and broad-spectrum antiproliferative activity against most of the tested cancer cell lines, with GI50 values in the range of 1.29–5.97 µM. DNA-flow cytometric analysis demonstrated the inhibition of cell proliferation by compound 6d at G1/S phase. Docking study showed that compound 6d interacts similarly to erlotinib and highlights the role of the halogen bond in enhancing the binding to the EGFR binding site for further developments.Click here for additional data file.
Authors: Matthew J Niederst; Haichuan Hu; Hillary E Mulvey; Elizabeth L Lockerman; Angel R Garcia; Zofia Piotrowska; Lecia V Sequist; Jeffrey A Engelman Journal: Clin Cancer Res Date: 2015-05-11 Impact factor: 12.531
Authors: P Skehan; R Storeng; D Scudiero; A Monks; J McMahon; D Vistica; J T Warren; H Bokesch; S Kenney; M R Boyd Journal: J Natl Cancer Inst Date: 1990-07-04 Impact factor: 13.506
Authors: Amaia Eleonora Maennling; Mehmet Kemal Tur; Marcus Niebert; Torsten Klockenbring; Felix Zeppernick; Stefan Gattenlöhner; Ivo Meinhold-Heerlein; Ahmad Fawzi Hussain Journal: Cancers (Basel) Date: 2019-11-20 Impact factor: 6.639
Figure 1.
Figure 2.
Scheme 1.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.