Synthesis, in vitro antitumour activity, and molecular docking study of novel 2-substituted mercapto-3-(3,4,5-trimethoxybenzyl)-4(3H)-quinazolinone analogues

Abstract A novel series of 2-substituted mercapto-3-(3,4,5-trimethoxybenzyl)-4(3H)-quinazolinones 1–20 was synthesised and evaluated for in vitro antitumour activity. N-(4-Chlorophenyl)-2-[(3-(3,4,5-trimethoxybenzyl)-4(3H)-quinazolinon-2-yl)thio)acetamide (7) and N-(3,4,5 trimethoxybenzyl)-2-[(3-(3,4,5-trimethoxybenzyl)-4(3H)-quinazolinon-2-yl)thio]propanamide (19) exhibited excellent antitumour properties, with mean growth inhibitory concentration (GI50) of 17.90 and 6.33 µΜ, respectively, compared with those of 5-fluorouracil 5-FU, gefitinib, and erlotinib (mean GI50: 18.60, 3.24, and 7.29 µΜ, respectively). Comparison of the GI50 (µM) values of compounds 7 and 19 versus those of 5-FU, gefitinib, and erlotinib against an in vitro subpanel of tumour cells lines showed that compounds 7 and 19 have activities almost equal to or higher than that of those standard drugs, especially against lung, CNS, and breast cancer cells. However, compounds 5, 10, 14, 15, 16, 17, and 20 exhibited effective antitumour activity against the different cell lines tested, with growth inhibition percentage (MGI%) of 19, 24, 19, 17, 16, 15, and 16, respectively. A modelling study was performed for compounds 7 and 19 by docking them into the EGFR kinase enzyme to study their mode of binding with the putative binding site.


X-ray crystallography
Data of compound 8 were collected on a Bruker APEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Mo Ka radiation, k ¼ 0.71073 Å at 296 (2) K. Cell refinement and data reduction were carried out by Bruker SAINT. SHELXT 35,36 was used to solve the structure. The final refinement was carried out by full-matrix least-squares techniques with anisotropic thermal data for non-hydrogen atoms on F. CCDC 1534954 contains the supplementary crystallographic data for this compound and can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif.

Antitumour screening
The antitumour evaluation was performed in nearly 60 human tumour cell lines obtained from nine organs, according to the rules of the Drug Evaluation Branch, NCI, Bethesda, MD 37-41 .

Docking methodology
All modelling experiments were conducted with MOE 2007.9 of the Chemical Computing Group Inc. (Montreal, Canada) 42,43 . The starting coordinates of the X-ray crystal structure of the EGFR   -

X-ray crystallography
The crystallographic data and refinement information of compound 8 are summarised in Tables S1-S3. The asymmetric unit is comprised of one independent molecule as shown in Figures S1 and S2. All the bond lengths and angles are in normal ranges 47 . In the crystal structure, the central quinazolin-4(3H)-one plane makes dihedral angles of 62.97 and 68.48 with the trimethoxybenzyl and flurophenyl groups, respectively, in different directions. The crystal packing was formed by three intermolecular interactions between N 3 ¼H 1 N 3 ···O 2 , C 9 ¼H 9 A···O 1 , and C 9 ¼H 9 B···O 2 with bond lengths 2.07 (3), 2.35, and 2.31 Å and bond angles 158(3) , 143 , and 144 , respectively.

Antitumour activity
Evaluation of the in vitro antitumour activity of the new synthesised compounds indicated in Table 1 was performed by the National Cancer Institute, Bethesda, MA. A single dose (10 mM) of the test compounds 2-20 was used in the full NCI 60 Human Tumor Cell Line Panel assay [37][38][39][40][41] .
The MGI% data revealed that compounds 7 and 19 were the most active, with antitumour activity against numerous cell lines belonging to diverse tumour subpanels (Table 1). Therefore, these compounds were tested against a panel of 57 tumour cell lines at a 5-log dose range [37][38][39][40][41] and the median growth inhibitory (GI 50 ), total growth inhibitory (TGI), and median lethal (LC 50 ) concentrations were calculated for each cell line ( Table 2).
Additionally, comparing the median GI 50 values (mM) of compounds 7 and 19 with those of 5-FU, gefitinib, and erlotinib against an in vitro subpanel of tumour cell lines showed that compounds 7 and 19 had activities almost equal to or higher than these known drugs against most cell lines (Table 3).

Molecular docking results
EGFR are tyrosine kinase enzymes that are overexpressed in numerous tumours such as colon, prostate, breast, ovarian, renal, and NSL cancers [31][32][33][34]48 . The inhibition of tyrosine kinase by quinazoline derivatives such as gefitinib and erlotinib ( Figure 1) is well documented 30,31 . Accordingly, the antitumour activity of the target compounds against colon, prostate, breast, ovarian, renal, and NSL cancers encouraged us to study the molecular docking of the compounds into the putative binding site on EGFR kinase. In this study, the most active compounds 7 (mean GI 50 : 17.90 mM) and 19 (mean GI 50 :6.33 mM) were docked into the putative active site of EGFR kinase, as well as the reference inhibitor erlotinib (mean GI 50 : 7.29 mM) 44 . All docking calculations were performed using MOE 2007.09 software (MOE of Chemical Computing Group Inc., Montreal, Canada) 42 .
The binding energies of the docked compounds 7, 19, and erlotinib (PDB code; 1M17) 44 into the putative binding site of EGFR were À22.11, À25.21, and À26.99 kcal/mol, respectively ( Figure 2). The molecular docking of the most active compound 19 revealed that it had similar orientation to erlotinib inside the receptor pocket, as well as additional bonding interactions. The docking results showed six typical and atypical hydrogen bonds with surrounding amino acids as shown in Figure 2. The trimethoxybenzyl fragment at C-3 of the quinazoline core formed bifurcated hydrogen bonds with amino acids Lys 721 . Moreover, the 4-quinazolinone ring uniquely formed two hydrogen bonds with the distinctive residues Met 769 and Thr 766 , similar to that observed in erlotinib ( Figure 2). Additionally, the carbonyl group of the acetanilide fragment of compound 19 formed bifurcated hydrogen bonds with the amino acid residue Cys 773 and Gly 772 augmenting the recognition within the enzyme binding site ( Figure 2 and Table 4).
Similar to compound 19, compound 7 binds with four hydrogen bonds. It was found that the trimethoxybenzyl group at C-3 of the quinazoline core was clearly recognised with hydrogen bonding to the amino acid residue Lys 721 similar to compound 19, while the quinazoline core was shifted away from the distinctive amino acid residue Met 769 (Figure 2). Additionally, two hydrogen bonds with the amino acid residue Gly 772 and the distinctive residue Thr 766 were found (Figure 2). It is obvious that the molecular docking results can be used to design novel quinazoline derivatives with potential binding to EGFR kinase and antitumour activity ( Table 4).

Conclusions
A novel series of 2-substituted mercapto-3-[3,4,5-trimethoxybenzyl]-4(3H)-quinazolinones 1-20, was synthesised and evaluated for in vitro antitumour activity. Compounds 7 and 19 showed strong antitumour activities with mean GI 50 values of 17.90 and 6.33 mM, TGI of 55.20 and 70.65 mM, and LC 50 of 80.52 and 93.58 mM; these values were compared with the reference drug 5-FU (GI 50 : 22.60 mM, TGI: 100 mM, and LC 50 : 100 mM). Comparing the median GI 50 (mM) of 5-FU, gefitinib, and erlotinib with that of compounds 7 and 19 showed that compounds 7 and 19 showed antitumour activities almost equal to or higher than that of the known drugs against most subpanel tumour cell lines. A molecular docking study for compounds 7 and 19 into the ATP binding site of EGFR-TK showed similar binding as that of erlotinib.

Disclosure statement
No potential conflict of interest was reported by the authors. Table 4. Results of the docking of compounds 7 and 19 into EGFR (pdb: 1m17), in comparison to the co-crystallised ligand (erlotinib).