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Full Paper Design, Synthesis, and Molecular Docking Studies of 2-(Furan-2-yl)quinazolin-4-one Derivatives as Potential Antiproliferative Agents Marwa F. Ahmed1* and Amany Belal2,3 1 2 3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo, Egypt Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Taif University, Taif, Kingdom of Saudi Arabia
Fifteen new derivatives of quinazolin-4-one bearing the 2-furyl moiety at position 2 and a substituted phenyl moiety at position 3 were designed and synthesized to be evaluated as cytotoxic agents. Their chemical structures were confirmed by spectral and elemental analysis; cytotoxic activity evaluation was performed against HEPG2, HCT116, and MCF7 cancer cell lines using the sulforhodamine-B assay. All the tested compounds except 6a showed high potency against the HEPG2 cancer cell line (IC50 8–101 nM/mL); 11 compounds out of 15 proved to be potent against HCT116 cells (IC50 3–49 nM/mL), also 11 of the tested compounds showed high potency against MCF7 cells with IC50 values ranging from 7 to 63 nM/mL. The rest of the tested compounds showed IC50 values of more than 100 nM/mL. Compounds 3e and 4d are the most active compounds against HEPG2 cells; in addition, 3e is the most active compound against MCF7 cells. Also, compounds 4a, 3a, and 3b are the most active compounds against HCT116 cells. Compounds 3a, 3b, 3e, 4a, and 4d were also evaluated for their inhibitory activity against the EGFR tyrosine kinase (EGFR-TK) and showed a percentage inhibitory activity ranging from 53 to 84%. The most potent EGFR-TK inhibitors, 3a (84%), 3b (75%), and 3e (60%), were docked into the ATP binding site of the EGFR to explore their binding mode and possible interactions. Keywords: Antitumor / Docking / EGFR-TKI / 2-Furyl / Quinazolinone / Tyrosine kinases Received: December 24, 2014; Revised: March 9, 2015; Accepted: March 20, 2015 DOI 10.1002/ardp.201400468
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Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction Quinazoline is a heterocyclic system of two fused six-membered aromatic rings, benzene and pyrimidine. Quinazolines have drawn more and more attention in the field of medicinal chemistryduetotheirsignificantanddifferentbiologicalactivities as antidiabetes [1], anticancer [2], antibacterial [3], anti-inflammation [4], antivirus [5], antipsychotic [6], and antiobesity [7].
Quinazolin-4-one is a heterocyclic scaffold occupying a unique role in the field of medicinal chemistry and represents an important scaffold for designing new bioactive anticancer agents [8]. 2,3-Disubstituted quinazolin-4(3H)-one I (Fig. 1) was reported to have good anticancer activity [9], in addition, compound II [8] showed a promising antitumor activity. 2-(2Thieno)-6-iodo-3-amino-3,4-dihydro-quinazolin-4-one III [10] exhibited a remarkable anticancer activity in comparison to the known drug 5-FU. Addition of lipophilic moiety at position 2 as furan-2-yl to quinazolin-4-one scaffold afforded the active anticancer agent IV [11], the previously reported
Correspondence: Dr. Amany Belal, Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt. E-mail: [email protected] Fax: 0020822317958
*Additional correspondence: Dr. Marwa F. Ahmed, E-mail: [email protected]
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O
CH3 N
O
NH2
N
N H
N O
N
Cl
I
II
Cl
O
O
I
NH
N
N
N S
N
O
N
IV
III
Figure 1. Anticancer active quinazolin-4-one derivatives.
work has proved that changing the para position of 3-phenyl moiety has a great impact on anticancer activity [12], as a result we synthesized the quinazolin-4-one scaffold having a lipophilic furan moiety at position 2 with various substituents at the 4 position of the aryl moiety to study these structural changes in correlation to the activity. It also was of our interest to substitute the 4 position of the 3-phenyl with biologically active anticancer moieties as chalcone [13], pyrazole [14], isoxazole [15], and pyrimidine [16].
O
O O
N
H2N
O
COCH3
N
COCH3
O
N
f usion
It has been noticed that epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of tumors [17]. It is found to be the most important target molecules to date [18]. Quinazolines are well-known inhibitors for these receptors and are, therefore, expected to have a great therapeutic potential in cancer treatment [19, 20]. Moreover, lapatinib, gefitinib, erlotinib, and canertinib are quinazoline derivatives that have proved to act as EGFR tyrosine kinase inhibitors [21]. All these facts encouraged us to investigate
2
1
ArCHO NaOH O a, Ar =
d, Ar =
b, Ar =
OMe
c, Ar =
CH 3
Ar
O
Cl
N
HO e, Ar =
N
O
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Scheme 1. Synthesis of compounds 3a–e.
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EGFR-TK inhibitory properties of the best active compounds of the newly designed quinazolines in this study. Furthermore, molecular docking studies were also performed to explore the binding mode and possible interactions with EGFR-TK active site.
Results and discussion Chemistry Compound 1 was synthesized according to the previously reported method [22]. Fusing compound 1 with p-aminoacetophenone at 160°C afforded 3-(4-acetylphenyl)-2(furan-2-yl)quinazolin-4(3H)-one 2 (Scheme 1), 1H NMR spectrum showed the appearance of additional aromatic protons and a singlet signal at 2.5 ppm indicating COCH3 protons. Claisen-Schmidt condensation of the acetyl derivative 2 with different aldehydes namely benzaldehyde, p-anisaldehyde, p-tolualdehyde, p-chlorobenzaldehyde, or 2-hydroxy benzaldehyde in ethanolic sodium hydroxide solution afforded the corresponding a,b-unsaturated ketones (chalcones) 3a–e, respectively (Scheme 1). Their 1H NMR spectra revealed the appearance of
additional aromatic protons and two duplets characterizing the two protons of chalcone moiety. Cyclocondensation of the unsaturated ketone 3a,b with hydrazine hydrate in absolute ethanol afforded the corresponding pyrazoline derivatives 4a,b. While cyclocondensation of the unsaturated ketone 3a,b with hydrazine hydrate in glacial acetic acid afforded the corresponding acetyl pyrazoline derivatives 4c,d (Scheme 2). 1H NMR spectra of 4a,b showed the appearance of CH2, CH, and NH signals indicating pyrazole moiety and that of compounds 4b,c showed signals for CH2, CH of pyrazole in addition to COCH3 signal. Moreover, their structures were also confirmed by mass, IR, 13 C NMR, and elemental analysis (Experimental section). Reacting a,b-unsaturated ketones 3a,b with hydroxylamine hydrochloride in ethanolic sodium hydroxide solution afforded the corresponding isoxazolines 5a,b, respectively, 1 H NMR revealed the appearance of CH2 and CH (isoxazoline protons). Cyclocondensation of the chalcones 3a,b with urea in the presence of HCl or with thiourea in the presence of NaOH afforded the corresponding tetrahydropyrimidinone 6a,b or tetrahydropyrimidine-thione derivatives 6c,d, respectively (Scheme 2). 1H NMR spectra of 6a–d revealed the appearance of CH2, CH, and NH signals characterizing the
O O
Ar N
O
N
thiourea
3a–e NH2NH2
NH2NH2
98%
EtOH
NH2OH
CH3COOH
N
O
Ar O
N
N Ar
N
O
H Ar
N
O
O
N
O 5a,b
4a–d a, Ar =
R=H
a, Ar =
b, Ar =
R=H
b, Ar =
OCH3
c, Ar = d, Ar =
X
N N
N
urea HCl
N O
R N
NaOH/EtOH
6a–d a, X = O, Ar = OCH3
R = COCH3 OCH3
R = COCH 3
b, X = O, Ar =
OCH3
c, X = S, Ar = d, X = S, Ar =
OCH3
Scheme 2. Synthesis of the target compounds 4a–d, 5a,b and 6a–d.
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tetrahydropyrimidine moiety, in addition IR, 13C NMR, mass, and elemental microanalysis were used as a tool in structures confirmation (Experimental section).
selective toward HCT116 and MCF7, respectively, than HEPG2 cell line.
SAR
Biological activity In vitro cytotoxic activity In the present work, the cytotoxic activity of the newly synthesized compounds was evaluated in vitro on liver (HEPG2), colon (HCT116), and breast (MCF7) cancer cell lines, using sulforhodamine-B (SRB) assay method and doxorubicin as a reference drug. Surviving fraction of cancer cells was plotted against drug concentration to obtain the survival curve. IC50 values (the dose causing 50% inhibition of viable cells) of the tested compounds were calculated and data are presented in Table 1 that shows the IC50 values for the tested compounds and doxorubicin in nM/mL. In general, the new compounds exhibited relevant cytotoxic activity, they showed to be active on HEPG2 except compound 6a, IC50 range of the active compounds is 8–101 nM/mL, and compounds 3e and 4d were the best active compounds with IC50 values equal to 8 and 9 nM/mL, respectively. The tested compounds, except 5a (its IC50 value exceeded 200 nM/mL), showed to be active on HCT116 cancer cell line with IC50 range from 3 to 150 nM/mL and compounds 3a, 3b, 3e, and 4a are the best active. Cytotoxic activity of the tested compounds against MCF7 was also promising as all of these compounds showed to be active, and compound 3e was the best active with IC50 value equal to 7 nM/mL. It is also noticed that some compounds showed cytotoxic selectivity toward specific cell lines, compound 3b showed to be 10 times more selective to HCT116 than HEPG2, compound 4a showed to be 20 times more selective on HCT116 than MCF7, finally compound 6a showed to be 100 and 50 times more
Substituting the para position of the 3-phenyl moiety with substituted chalcones afforded compounds 3a–e with an important cytotoxic activity on HEPG2, HCT116, and MCF7 cell lines, 4-chalcone moiety bearing 2-hydroxy phenyl was the best active and broad spectrum compound on the tested cancer cell lines, its IC50 ranges from 7 to 10 nM/mL. However, substituting the 4-chalcone moiety with 4-methylphenyl 3c or 4-chlorophenyl 3d leads to a decrease in activity on all the tested cell lines. Substituting the 3-phenyl moiety with chalcone bearing p-methoxyphenyl 3b or p-methylphenyl 3c leads to an increased activity and selectivity on colon cancer cell line HCT116. Substituting the para position of 3-phenyl moiety with pyrazole afforded compounds 4a–d with good cytotoxic activity, compound 4a has a 5-phenyl-1H-pyrazol-3-yl at the para position of 3-phenyl showed to be the best active on colon cancer cells, and compound 4d that has 1-acetyl-5-(4methoxyphenyl)-1H-pyrazol-3-yl at the 4 position of the 3-phenyl showed a highly potent activity on liver cancer cells (IC50 ¼ 9 nM/mL). Substituting the 3-phenyl with isoxazole moiety 5a,b leads to a decrease in activity; however, incorporating a 4-methoxyphenyl at the 5 position of isoxazol-3-yl moiety 5b gave a potent activity against HEPG2. Substituting the 3-phenyl with pyrimidine moiety 6a–d leads to good cytotoxic activity and the 2-thioxopyrimidines 6c,d were found to be more active than 2-oxopyrimidines 6a,b on HEPG2 and HCT116 cancer cell lines; however, the oxo derivatives 6a,b were found to be more active than the thioxo 6c,d on breast cancer cell line.
Table 1. In vitro anticancer activity of the newly synthesized quinazolines on liver (HEPG2), colon (HCT116), and breast (MCF7) cancer cell lines. IC50 (nM/mL SD) Comp. no. 3a 3b 3c 3d 3e 4a 4b 4c 4d 5a 5b 6a 6b 6c 6d Dox.
HEPG2 16.35 73.31 50.95 19.12 8.31 101.07 21.76 39.74 8.51 68.82 13.65 1781.13 50.74 166.6 19.09 5.66
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0.89 0.25 0.24 0.3 0.29 0.39 0.29 0.34 0.2 0.36 0.28 0.53 0.31 0.4 0.39 0.10
HCT116 7.67 6.63 49.24 38.04 9.73 2.86 122.18 113.74 22.51 223.78 150.4 11.04 40.38 10.24 15.47 4.79
0.97 0.18 0.19 0.15 0.23 0.43 0.49 0.44 0.37 0.35 0.3 0.29 0.21 0.37 0.36 0.45
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MCF7 42.42 123.27 111.2 15.71 6.49 55.31 43.38 29 30.70 115.75 114.22 24.81 15.76 29.62 62.98 4.44
0.63 0.24 0.23 0.13 0.28 0.37 0.38 0.23 0.3 0.26 0.42 0.41 0.39 0.41 0.36 0.11
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Table 2. EGFR tyrosine kinase assay of the best active quinazoline derivatives at 10 mM. Comp. no. 3a 3b 3e 4a 4d Erlotinib
% inhibition of EGFR tyrosine kinase 84 75 60 53 58 100
EGFR inhibition In this study, the best active compound on MCF7 (3e), the best active compounds on HEPG2 (3e and 4d), and the best active compounds on HCT116 (4a, 3a, and 3b) were screened at 10 mM for their inhibitory activity against EGFR-TK. The inhibitory range of the tested compounds is 53–84%, compound 3a is the most potent one, its inhibitory percent was at the value of 84%. Moreover, compounds 3a, 3b, and 3e that have chalcone substituted moiety attached to the para position of the 3-phenyl ring showed to be more potent inhibitors than derivatives bearing a pyrazole moiety 4a,d at the same position. The inhibitory percentage of chalcone
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substituted derivatives 3a, 3b, and 3e is 60–84%; however, the inhibitory percentage of pyrazole substituted derivatives 4a and 4d is 53–58% (Table 2). From the obtained data, we can say that the best active cytotoxic quinazolines 3a,b,e and 4a,d exhibited moderate to good activity against EGFR-TK. Compounds 3a and 3b, which showed a very good cytotoxic activity on HCT116 cell line, revealed inhibitory percentage against EGFR-TK equaling 84 and 75%, respectively; therefore, these two compounds can be considered as new leads for developing effective inhibitors against EGFR-TK. The other compounds 3e and 4a,d, which showed a good cytotoxic activity but had moderate (53–60%) inhibitory percentage against EGFR-TK, may need further exploration to detect their molecular targets.
Molecular docking studies The best active inhibitors of EGFR-TK, 3a, 3b, and 3e, were docked into the ATP binding site of EGFR to explore their binding mode and possible interactions with this receptor. All the calculations were performed using Molecular Operating Environment software (MOE) provided by the Chemical Computing Group, Canada, installed on Intel core i5, 2.5 G. EGFR cocrystallized with erlotinib (pdb: 1M17) [23] is the protein data bank file used in performing the docking studies. Docking was performed using London dG force and
Figure 2. 2D interactions of compound 3a with the ATP binding site of the EGFR.
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Figure 3. 2D interactions of compound 3b with the ATP binding site of the EGFR.
refinement of the results was done using Force field energy. Validation process was performed by docking the cocrystallized ligand (erlotinib) into ATP binding site of EGFR-TK to study the scoring energy (S), root mean standard deviation (rmsd), and amino acid interactions. Erlotinib is fitted into the active site pocket with S ¼ 21.66 kcal/mol and rsmd ¼ 1.4682, it also showed hydrogen bonding with Met 769 and another hydrogen bonding interaction with Thr 766 mediated by a molecule of water. The selected compounds were docked into ATP binding site of EGFR, they showed good fitting into the binding site with docking score energy equal to 20.93 (3a), 18.88 (3b), and 17.97 (3e). Moreover, compound 3a showed a hydrogen bonding with Cys 773 and Lys 721 amino acids through the CO of chalcone moiety and N-1 of quinazolinone nucleus, respectively, in addition to arene–arene interaction between the furyl moiety and Phe 699 amino acid (Fig. 2). Compound 3b showed two hydrogen bonds with Cys 773 and Thr 766 amino acids through CO of the quinazolinone and O of the methoxy group, respectively (Fig. 3). Compound 3e showed only one hydrogen bond with Asp 851 through H of the 2-hydroxyl group (Fig. 4). The 3D molecular surface map of the most
From this study, we can conclude that quinazolin-4-one bearing a 2-furyl moiety at position 2 and a phenyl moiety at position 3 proved to be an important scaffold for designing new cytotoxic active agents against HEPG2, HCT116, and MCF7 cell lines. Substituting the para position of 3-phenyl moiety in this scaffold with chalcone bearing 2-hydroxyphenyl revealed a broad spectrum anticancer agent 3e against the three cancer cell lines and substituting it with 4-methoxyphenyl 3b or 4-phenyl 3a afforded highly potent and selective compounds toward colon cancer cell line HCT116. However, 4-para chlorophenyl 3d and 4-methylphenyl 3c substituents lead to decrease in the activity. Substituting the para position of 3-phenyl ring with pyrazole leads to more active compounds than isoxazole and pyrimidine substituents. Compounds bearing a chalcone and pyrazole moiety at the para position of 3-phenyl ring 3a,b,e and 4a,d showed to act as good
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active compounds 3a, 3b, and 3e docking into the EGFR binding site is shown in Fig. 5.
Conclusion
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Figure 4. 2D interactions of compound 3e with the ATP binding site of the EGFR.
inhibitors for EGFR-TK. Chalcone derivatives 3a,b,e were found to be more potent inhibitors than pyrazole derivatives 4a,d. This work helped in finding potent inhibitors of EGFR belonging to new chemical sub-classes of quinazolinones (3a,b) and this opens the opportunity to expand further drug discovery projects. Molecular docking studies helped in understanding the various interactions between the ligands
and EGFR binding site in detail and this may help in design of novel potent EGFR-TK inhibitors.
Experimental Chemistry All melting points are uncorrected, elemental analyses were carried out in the microanalytical unit of National Research Centre and Cairo University, Egypt. IR spectra were recorded on FT-IR spectrophotometer Nexus 670Nicolet (USA) and Perkin Elmer-9712 spectrophotometer (Waltham, MA, USA). 1H NMR spectra were determined on a Varian-Gemini-300 MHz and Jeol-Ex 270 MHz NMR spectrometer using TMS as an internal standard. 13C NMR (DMSO-d6) spectra were recorded at 100.62 MHz at the aforementioned research center in Cairo University. Mass spectra were determined on Finnigan Mat SSQ 7000, mode EI 70 eV (Thermo Instrument Systems, Inc., USA). Thin layer chromatography was carried out on silica gel 60 F254 (Merck) plates using chloroform/petroleum ether/methanol (7:4:1) as an eluent system.
3-(4-Acetylphenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (2) Figure 5. 3D molecular surface map showing the docked poses of the most active compounds 3a (green), 3b (red), and 3e (blue) into EGFR binding site (PDB ID: 1M17).
A mixture of the benzoxazine 1 (2.13 g, 0.01 mol) and p-aminoacetophenone (1.35 g, 0.01 mol) was heated together upon fusion at 160°C on sand bath for 2 h. After cooling the crude mass was crystallized from ethanol twice to give dark
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brown crystals of compound 2, m.p. 250°C, 80% yield. Analysis calculated for C20H14N2O3 (330.34); calcd.: % C, 72.72; H, 4.27; N, 8.48, found: % C, 72.52; H, 4.17; N, 8.40. IR: ymax/cm1 3390 (C–H aromatic), 1700 (C –– O of acetyl), 1680 (C –– O of quinazolinone), 1633 (C –– N), and at 1590 (C –– C). 1H NMR (DMSO-d6, ppm): d 2.5 (3H, s, COCH3), 6.5–8.1 (m, 11H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 197, 164, 160.6, 144.5, 143, 141.7, 137.1, 136.6, 133.4, 129, 127.3, 124.3, 120.7, 109.9, 109.4, 26.6. MS (m/z, R.I.): calcd. for C20H14N2O3: 330.10; found: 330.
General method for the preparation of 3a–e A mixture of the ketone 2 (0.002 mol) and the appropriate aromatic aldehyde (0.002 mol) in ethanol (10 mL) was prepared. 5% NaOH in ethyl alcohol (10 mL) was added dropwise within 15 min. The reaction mixture was refluxed for 3 h then cooled and the crude precipitated material was filtered off, air dried, and then crystallized from the proper solvent to give the chalcones 3a–e, respectively.
(E)-3-(4-Cinnamoylphenyl)-2-(furan-2-yl)quinazolin-4(3H)one (3a) Crystallized from ethanol to give yellow crystals, m.p. 155°C in 70% yield. Analysis for C27H18 N2O3 (418.44), calcd. %C, 77.50; H, 4.34; N, 6.69; found: %C, 77.45; H, 4.30; N, 6.62; IR: ymax/cm1 1690 (C –– O quinazolinone), 1665 (C –– O of the a,bunsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSO-d6, ppm): 6.6–6.8 (2H, d, d, CH –– CH) and 6.9–7.9 (m, 16H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 189.7, 165, 160.6, 145.2, 144.5, 142.7, 140.5, 139.5, 135.2, 134.5, 134.2, 131.4, 129.7, 129.5, 127.9, 127.2, 125.9, 125.4, 124.9, 121.3, 120.6, 109.9, 109.4. MS (m/z, R.I.): calcd. for C27H18N2O3: 418.13; found: 418.13.
(E)-2-(Furan-2-yl)-3-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)quinazolin-4(3H)-one (3b) Crystallized from glacial acetic acid to give yellow crystals, m.p. 190°C in 75% yield. Analysis for C28H20N2O4 (448.47), calcd. % C, 74.99; H, 4.50; N, 6.25; found: %C, 74.97; H, 4.49; N, 6.24; IR: ymax/cm1 1700 (C –– O quinazolinone), 1670 (C –– O of the a,bunsaturated ketone), and at 1600 (C –– N). 1H NMR spectrum (DMSO-d6, ppm): 3.80 (3H, s, OCH3), 6.8–6.9 (2H, d, d, CH –– CH) and 6.9–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 190.4, 166.7, 162.4, 159.9, 146.2, 145.9, 143.4, 141.7, 140.5, 133.5, 133.2, 131.4, 130.7, 127.7, 127.5, 126.6, 124.9, 124.3, 120.9, 114.2, 112.7, 112.4, 59.7. MS (m/z, R.I.): calcd. for C28H20N2O4: 448.14; found: 448.14.
(E)-2-(Furan-2-yl)-3-(4-(3-p-tolylacryloyl)phenyl)quinazolin-4(3H)-one (3c) Crystallized from glacial acetic acid to give yellow crystals, m.p. 240°C in 70% yield. Analysis for C28H20 N2O3 (432.47), calcd. %C, 77.76; H, 4.66; N, 6.48; found: %C, 77.72; H, 4.64; N, 6.46; IR: ymax/cm1 1700 (C –– O quinazolinone), 1665 (C –– O of the a,bunsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSO-d6, ppm): 2.5 (3H, s, CH3), 6.7–6.9 (2H, d, d, CH –– CH) and 6. 8–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 188.5, 165.3,
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161.2, 145.1, 144.9, 143.2, 140.6, 137.6, 134.5, 134.2, 132.2, 131.9, 128.9, 128.5, 127.3, 126.7, 125.7, 122.3, 120. 8, 111.5, 111, 22.3. MS (m/z, R.I.): calcd. for C28H20N2O3: 432.15; found: 432.15.
(E)-3-(4-(3-(4-Chlorophenyl)acryloyl)phenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (3d) Crystallized from glacial acetic acid to give yellow crystals, m.p. 250°C in 70% yield. Analysis for C27H17ClN2O3 (452.89), calcd. %C, 71.60; H, 3.78; N, 6.19, found: %C,71.59; H, 3.69; N, 6.17. IR: ymax/cm1 1710 (C –– O quinazolinone), 1670 (C –– O of the a,b-unsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSOd6, ppm): 6.7–6.9 (2H, d, d, CH –– CH) and 6. 8–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 190.2, 165.7, 161.9, 146.1, 143.9, 141.7, 138.5, 135.6, 135.5, 135.2, 131.4, 129, 128.7, 127.9, 125.6, 125.2, 124.9, 122.3, 120.9, 109.5, 109. MS (m/z, R.I.): calcd. for C27H17ClN2O3: 452.09; found: 452.09.
(E)-2-(Furan-2-yl)-3-(4-(3-(2-hydroxyphenyl)acryloyl)phenyl)quinazolin-4(3H)-one (3e) Crystallized from ethanol to give brown crystals, m.p. 159°C in 70% yield. Analysis for C27H18N2O4 (434.44), calcd. %C, 74.64; H, 4.18; N, 6.45; found: %C, 74.60; H, 4.11; N, 6.43; IR: ymax/cm1 3380 (OH), 1720 (C –– O quinazolinone), 1675 (C –– O of the a,b-unsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSO-d6, ppm): 6.7–6.9 (2H, d, d, CH –– CH), 6. 8–8.2 (m, 15H, Ar-H, and furan-H) and 11.5 (s, 1H, O–H). 13C NMR (DMSO-d6): 188.9, 166.7, 160.7, 157.1, 145.2, 143, 142.4, 141, 138.9, 133.5, 133.3, 131.4, 129.3, 128.9, 127.3, 126.7, 126.5, 124.9, 122.6, 121.9, 121.3, 120.7, 117.6, 109.7, 109.2. MS (m/z, R.I.): calcd. for C27H18N2O4: 434.13; found: 434.13.
General method for the preparation of 4a,b A mixture of the chalcone 3a,b (0.005 mol) and hydrazine hydrate (2.5 mL, 0.005 mol, 98%) in abs. ethanol (25 mL) was heated under reflux for 10 h. After cooling, the separated material was filtered off, air dried, and then crystallized from the proper solvent to give 4a,b, respectively.
2-(Furan-2-yl)-3-(4-(5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)quinazolin-4(3H)-one (4a) Crystallized from ethanol to give yellow crystals, m.p. 200°C in 70% yield. Analysis for C27H20N4O2 (432.47), calcd. %C, 74.92; H, 4.54; N, 12.90, found: %C, 74.98; H, 4.66; N, 12.95; IR: ymax/cm1 3385 (NH), 3030 (CH, aromatic), 1670 (C –– O), and 1635 (C –– N). (DMSO-d6, d ppm): 3.3 (d,d, 2H, CH2, pyrazoline ring, J ¼ 5.6 Hz), 3.90 (t, 1H, CH of pyrazoline, 7.21 (s, NH, exchangeable with D2O), 6. 8–8.2 (m, 16H, Ar-H, and furan-H). 13 C NMR (DMSO-d6) d ppm: 164.3, 161.7, 151.7, 144.5, 143.5, 141.7, 135.9, 133.4, 132, 129.4, 128.5, 127.3, 126.9, 126.7, 126.6, 124.5, 120.6, 121.9, 110.5, 110.2, 49.1, 42.6. MS (m/z, R.I.): calcd. for C27H20N4O2: 432.16; found: 432.16.
2-(Furan-2-yl)-3-(4-(5-(4-methoxyphenyl)-4,5-dihydro-1Hpyrazol-3-yl)phenyl)quinazolin-4(3H)-one (4b) Crystallized from ethanol to give brown crystals, m.p. 220°C in 70% yield. Analysis for C28H22N4O3 (462.50), calcd. %C,
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72.71; H, 4.79; N, 12.11; found: %C, 72.65; H, 4.75; N, 12.00; IR: ymax/cm1 3390 (NH), 3035 (CH, aromatic), 1670 (C –– O), and 1640 (C –– N). 1H NMR (DMSO-d6, ppm) 3.4 (d,d, 2H, CH2, pyrazoline ring, J ¼ 5.6 Hz), 3.70 (3H, s, OCH3), 3.90 (t, 1H, CH of pyrazoline), 7.4 (s, NH, exchangeable with D2O), 6.9–8.3 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 166.2, 162.4, 158.6, 152.6, 145.7, 144.2, 143.7, 135.8, 135.4, 133.4, 132, 130.2, 127.3, 126.7, 126.5, 125.4, 120.8, 114.1, 109.9, 109.2, 55.8, 50.2, 43.9. MS (m/z, R.I.): calcd. for C28H22N4O3: 462.17; found: 462.17.
General method for the preparation of 4c,d A mixture of the chalcone 3a,b (0.005 mol) and hydrazine hydrate (2.5 mL, 0.005 mol, 98%) in the presence of (10 mL) glacial acetic acid was heated under reflux for 10 h. After cooling, the mixture was poured into ice/water and filtered off, air dried, and then crystallized from the proper solvent to give 4c,d, respectively.
3-(4-(1-Acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (4c) Crystallized from ethanol to give dark brown crystals, m.p. 192°C in 70% yield. Analysis for C29H22N4O3 (474.51), calcd. % C, 73.40; H, 4.67; N, 11.81; found: %C, 73.35; H, 4.64; N, 11.80; IR: ymax/cm1 3030 (CH, aromatic), 1690 (C –– O quinazolinone, 1670 (C –– O, acetyl), and 1635 (C –– N). 1H NMR (DMSO-d6, ppm) 2.1 (3H, s, COCH3), 3.3 (d, d, 2H, CH2, pyrazoline ring), 3.90 (t, 1H, CH of pyrazoline, 6.8–8.2 (m, 16H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 168.5, 164.9, 160.6, 151.6, 144.3, 143.4, 141.9, 135.2, 135.9, 129.2, 128.5, 127.6, 126.9, 126.6, 126.4, 124.4, 123.7, 112.7, 112.2, 58.9, 39.3, 23.4. MS (m/z, R.I.): calcd. for C29H22N4O3: 474.17; found: 474.17.
3-(4-(1-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol3-yl)phenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (4d) Crystallized from ethanol to give brown crystals, m.p. 149°C in 65% yield. Analysis for C30H24N4O4 (504.54), calcd. %C, 71.42; H, 4.79; N, 11.10; found: %C, 71.40; H, 4.75; N, 11.95. IR: ymax/cm1 3035 (CH, aromatic), 1695 (C –– O quinazolinone, 1670 (C –– O, acetyl), and 1640 (C –– N). 1H NMR (DMSO-d6, ppm) 2.2 (3H, s, COCH3), 3.4 (d, d, 2H, CH2, pyrazoline ring), 3.70 (3H, s, OCH3), 3.95 (t, 1H, CH of pyrazoline), 6.9–8.3 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 169.4, 165.9, 162.6, 158.6, 151.4, 144.5, 143, 140.4, 136, 134, 133.2, 132, 130.2, 127.6, 127.3, 126.6, 124.5, 121.7, 114.1, 109.2, 109, 58.9, 56.9, 39.3, 23.4. MS (m/z, R.I.): calcd. for C30H24N4O4: 504.18; found: 504.18.
General method for the preparation of 5a,b A mixture of chalcone 3a,b (3 mmol) and hydroxylamine hydrochloride (5 mmol) in sodium hydroxide solution (0.5 g NaOH in 0.5 mL water) in ethanol (60 mL) was refluxed for 3 h. The product obtained upon cooling was filtered off, washed with water, and recrystallized from the proper solvents to obtain the desired compounds 5a,b, respectively.
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2-(Furan-2-yl)-3-(4-(5-phenyl-4,5-dihydroisoxazol-3-yl)phenyl)quinazolin-4(3H)-one (5a) Crystallized from ethanol to give brown crystals, m.p. 235°C in 65% yield. Analysis for C27H19N3O3 (433.46), calcd. %C, 74.81; H, 4.42; N, 9.69; found: %C, 74.79; H, 4.40; N, 9.64. IR: ymax/cm1 3030 (CH, aromatic), 1665 (C –– O), and 1630 (C –– N). 1 H NMR (DMSO-d6, d ppm): 3.6–3.72 (d, d, 2H, CH2, isoxazoline ring), 6.24 (t, 1H, CH, isoxazoline ring), 6.5–8.1 (m, 16H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 164.4, 160.9, 156.2, 143.9, 143.3, 142.4, 141.6, 135, 133.5, 130.5, 129.4, 130.2, 128.6, 128.3, 128.1, 126.7, 126.5, 126, 124.7, 120.8, 112.7, 112.4, 82.8, 42. MS (m/z, R.I.): calcd. for C27H19N3O3: 433.14; found: 433.14.
2-(Furan-2-yl)-3-(4-(5-(4-methoxyphenyl)-4,5dihydroisoxazol-3-yl)phenyl)quinazolin-4(3H)-one (5b) Crystallized from glacial acetic acid to give brown crystals, m.p. 155°C in 65% yield. Analysis for C28H21N3O4 (463.48), calcd. % C, 72.56; H, 4.57; N, 9.07; found: %C, 72.51; H, 4.52; N, 9.03. IR: ymax/cm1 3040 (CH, aromatic), 1670 (C –– O), and 1630 (C –– N). 1 H NMR (DMSO-d6, d ppm): 3.7–3.8 (d, d, 2H, CH2, isoxazoline ring), 3.9 (3H, s, OCH3), 6.24 (t, 1H, CH, isoxazoline ring and 6.7–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 165, 160.4, 159.4, 156.6, 144.9, 143.2, 142.4, 134.7, 133.4, 132, 129.6, 127.3, 127, 125.9, 125.7, 125, 124.5 120.8, 114.5, 110.7, 109.2, 82.8, 55.8, 42. MS (m/z, R.I.): calcd. for C28H21N3O4: 463.15; found 463.15.
General method for the preparation of 6a,b A mixture of the chalcone 3a,b (0.005 mol) and urea (0.5 g, 0.005 mol) in ethanol (20 mL) and conc. HCl (5 mL) was refluxed for 7 h. The reaction mixture was concentrated to half its volume, cooled, and neutralized with NH4OH solution. The precipitated solid was filtered off, washed with water, air dried, and crystallized from the proper solvent to give compound 6a,b.
2-(Furan-2-yl)-3-(4-(2-oxo-6-phenyl-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6a) Crystallized from glacial acetic acid to give brown crystals, m.p. 205°C in 60% yield. Analysis for C28H20N4O3 (460.48), calcd. % C, 73.03; H, 4.38; N, 12.17; found: %C, 73.00; H, 4.34; N, 12.11. IR: ymax/cm1 3215–3600 (OH enolic of pyrimidine), 3040 (CH, aromatic), 1690 (C –– O), and 1630 (C –– N). 1H NMR (DMSO-d6, d ppm): 3.4 (2H, d, CH2 of pyrimidinone), 5.6 (1H, t, CH of pyrimidinone), and 6.4–8.1 (m, 17H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 164.6, 164, 163, 160.6, 144.5, 143.5, 143, 141.7, 136.2, 133.5, 129.4, 128.5, 127.3, 126.9, 126.7, 126.6, 124.5 121.2, 109.9, 109.4, 43.6, 42.7. MS (m/z, R.I.): calcd. for C28H20N4O3: 460.15; found: 460.15.
2-(Furan-2-yl)-3-(4-(6-(4-methoxyphenyl)-2-oxo-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6b) Crystallized from ethanol to give brown crystals, m.p. 150°C in 60% yield. Analysis for C29H22N4O4 (490.51), calcd. %C, 71.01; H, 4.52; N, 11.42; found: %C, 71.94; H, 4.49; N, 11.40; IR:
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ymax/cm1 3220–3610 (OH enolic of pyrimidine), 3055 CH, aromatic), 1690 (C –– O), and 1635 (C –– N). 1H NMR (DMSO-d6, d ppm): 3.5 (2H, d, CH2 of pyrimidinone), 3.9 (3H, s, OCH3), 5.7 (1H, t, CH of pyrimidinone), and 6.4–8.4 (m, 16H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 166.2, 165, 164.5, 162.3, 158.6, 145.2, 143.3, 142.9, 137.5, 135.8, 135, 133.4, 129.9, 127.3, 126.7, 126.6, 125.2, 120.8, 114.1, 113.3, 113, 56.8, 43.9, 40.5. MS (m/z, R.I.): calcd. for C29H22N4O4: 490.16; found: 490.16.
General method for the preparation of 6c,d A mixture of the chalcone 3a,b (0.005 mol) and thiourea (0.005 mol) in the presence of 0.5 g of NaOH in 5 mL of water. The mixture was refluxed in (25 mL) of ethanol for 6 h then concentrated under vacuum and neutralized with diluted HCl. The precipitated material was filtered off, washed with water, dried, and crystallized from ethanol to give 6c,d.
2-(Furan-2-yl)-3-(4-(6-phenyl-2-thioxo-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6c) Crystallized from ethanol to give brown crystals, m.p. 270°C in 60% yield. Analysis for C28H20N4O2S (476.55), calcd. %C, 70.57; H, 4.23; N, 11.76; found: %C, 70.51; H, 4.20; N, 11.73; IR: ymax/cm1 3210–3590 (OH enolic of pyrimidine), 3030 (CH, aromatic), 1690 (C –– O), 1630 (C –– N), and 1270 (C –– S). 1H NMR (DMSO-d6, d ppm): at 3.5 (2H, d, CH2 of pyrimidinone), 5.7 (1H, t, CH of pyrimidinone), and 6.5–8.1 (m, 17H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 190.2, 164.5, 165, 160.4, 158.9, 146.7, 145, 143, 143.4, 139.2, 137.6, 135.7, 133.6, 130.5, 129.4, 128.7, 128.5, 126.5, 123.9, 114.9, 114.2, 57.8, 53.7, 43.5. MS (m/z, R.I.): calcd. for C28H20N4O2S: 476.13; found: 476.13.
2-(Furan-2-yl)-3-(4-(6-(4-methoxyphenyl)-2-thioxo-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6d) Crystallized from ethanol to give brown crystals, m.p. 215°C in 60% yield. Analysis for C29H22N4O3S (506.57), calcd. %C, 68.76; H, 4.38; N, 11.06; found: %C, 68.71; H, 4.35; N, 11.04; IR: ymax/cm1 3215–3590 (OH enolic of pyrimidine), 3035 (CH, aromatic), 1690 (C –– O), 1630 (C –– N), and 1275 (C –– S). 1H NMR (DMSO-d6, d ppm): at 3.5 (2H, d, CH2 of pyrimidinone), 3.8 (3H, s, OCH3), 5.7 (1H, t, CH of pyrimidinone), 6.5–8.1 (m, 16H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 189, 167.4, 166.1, 161.6, 144.2, 143.7, 143, 141.9, 136.2, 135, 132.7, 129.4, 128.4, 127.9, 126.9, 126.7, 124.5, 122.9, 111.9, 111.2, 52, 42.9. MS (m/z, R.I.): calcd. for C29H22N4O3S: 506.14; found: 506.14.
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University, Cairo, Egypt). Reagents and chemicals were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). Cells were seeded for 24 h in a 96-well microtiter plates at a concentration of 1000–2000 cells/well, 100 mL/well, then cells were incubated for 48 h with various concentrations (0, 6.25, 12.5, 25, 50, and 100 mg/mL) of the tested compounds and doxorubicin, three wells were used for each concentration; after incubation for 48 h, the cells were fixed with 10% trichloroacetic acid 150 mL/well for 1 h at 4°C, washed by distilled water for three times. Wells were stained for 10–30 min at room temperature with 0.4% SRB, dissolved in 1% acetic acid 70 mL/well. Washed with acetic acid 1% to remove unbound dye till colorless drainage obtained. The plates were subjected to air drying, 24 h not exposed to UV. The dye was solubilized with 150 mL/well of 10 mM Tris-EDTA (pH 7.4) for 5 min on a shaker at 1600 rpm. The optical density of each well was measured spectrophotometrically at 545 nm with an ELISA microplate reader. The percentage of surviving cells was calculated and plotted against different concentrations of the tested compounds to obtain the survival curve. The IC50 values were calculated using sigmoidal concentration–response curve fitting models (Sigmaplot software).
EGFR TK inhibition assay Compounds 3a, 3b, 3e, 4a, and 4d were tested in vitro for inhibition of EGFR tyrosine kinase using Kinase-Glo Plus luminescence kinase assay kit. The tested compounds were dissolved in DMSO. They were then added to reaction plates containing the EGFR tyrosine kinase in assay buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 10 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 0.02% Brij35, 0.02 mg/mL bovine serum albumin, 0.1 mM Na3VO4, 2 mM dithiothreitol, 1% DMSO). Reactions were initiated by addition of a mixture of ATP (Sigma–Aldrich) and 33P ATP (Perkin Elmer) to a final concentration of 10 mM. Reactions were carried out at room temperature for 2 h, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman, Inc., Piscataway, NJ, USA). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid [25]. The results are presented as percentage enzyme inhibition in comparison to erlotinib as a reference EGFR-TK inhibitor.
Molecular docking
Cytotoxic activity of the newly synthesized compounds was evaluated using the SRB assay [24]. HEPG2, HCT116, and MCF7 cancer cell lines were obtained from the American Type Culture Collection (ATCC, MN, USA) through the Tissue Culture Unit (The Egyptian Organization for Biological Products and Vaccines, Vacsera, Egypt). Cytotoxicity was performed at the Center for Genetic Engineering (Al-Azhar
Molecular modeling studies were carried out on an Intel Core i5, 2.53 GHz processor, 4 GB RAM memory with Windows XP 32-bit operating system using Molecular Operating Environment software. Energy minimization was performed with MOE, RMSD gradient of 0.05 kcal/mol, MMFF94X Force field and the partial charges were automatically calculated. The X-ray crystallographic structure of erlotinib cocrystallized with EGFR was obtained from the protein data bank (PDB ID: 1M17). The ATP binding site of EGFR was prepared for docking studies where erlotinib was removed from the active site; hydrogen atoms were added to the structure with their standard geometry; MOE Alpha Site Finder was used for the
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active sites detection; the obtained model was saved as moe file then used in predicting interactions at the active site between the selected compounds and EGFR; and the 2D structures of the docked compounds were generated by ChemDraw, transformed to their 3D by Moe program, protonated, and subjected to energy minimization, then saved as mdb file to be docked into the active site of EGFR. The authors have declared no conflict of interest.
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[10] A. M. Al-Obaid, S. G. Abdel-Hamide, H. A. El-Kashef, A. A. M. Abdel-Aziz, H. A. Al-Khamees, H. I. El-Subbagh, Eur. J. Med. Chem. 2009, 44, 2379–2391. [11] M. N. Noolvi, H. M. Patel, V. Bhardwaj, A. Chauhan, Eur. J. Med. Chem. 2011, 46, 2327–2346. [12] M. F. Ahmed, M. Youns, Arch. Pharm. Chem. Life Sci. 2013, 346, 1–8. [13] S. Shukla, R. S. Srivastava, A. Sodhi, K. Pankaj, Med. Chem. Res. 2013, 22, 1604–1617. [14] T. Taj, R. R. Kamble, T. M. Gireesh, R. K. Hunnur, S. B. Margankop, Eur. J. Med. Chem. 2011, 46, 4366–4373. [15] P. Palanisamy, S. J. Jenniefer, P. T. Muthiahb, S. Kumaresan, RSC Adv. 2013, 42(3), 19300–19310. [16] S. Cesarini, A. Spallarossa, A. Ranise, S. Schenone, C. Rosano, P. La Colla, G. Sanna, B. Busonera, R. Loddo, Eur. J. Med. Chem. 2009, 46, 1106–1118. [17] J. M. Useros, J. G. Foncillas, Oral Oncol. 2015, 51, 423– 430. [18] J. Wang, J. M. Yu, S. W. Jing, Y. Guo, Y. J. Wu, N. Li, W. P. Jiao, L. Wang, Y. J. Zhang, Asian Pac. J. Cancer Prev. 2014, 15(14), 5889–5893. [19] K. N. Ganjoo, H. Wakelee, Biol. Targets Ther. 2007, 1(4), 335–346. [20] S. Dhillon, A. J. Wagstaff, Drugs 2007, 67(14), 2109–2110. [21] J. Fricker, Lancet Oncol. 2006, 7, 621. [22] M. N. Noolvi, H. M. Patel, V. Bhardwaj, A. Chauhan, Eur. J. Med. Chem. 2011, 46, 2327–2346. [23] http://www.rcsb.org/pdb/explore.do?pdbId¼1m17. Last accessed on Nov. 27, 2014. [24] P. Skehan, R. Storeng, D. Scudiero, A. Monks, I. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney, M. R. Boyd, J. Nat. Cancer Inst. 1990, 82(13), 1107–1112. [25] H. Ma, S. Deacon, K. Horiuchi, Expert Opin. Drug Discov. 2008, 3, 607–621.
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Full Paper Design, Synthesis, and Molecular Docking Studies of 2-(Furan-2-yl)quinazolin-4-one Derivatives as Potential Antiproliferative Agents Marwa F. Ahmed1* and Amany Belal2,3 1 2 3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo, Egypt Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Taif University, Taif, Kingdom of Saudi Arabia
Fifteen new derivatives of quinazolin-4-one bearing the 2-furyl moiety at position 2 and a substituted phenyl moiety at position 3 were designed and synthesized to be evaluated as cytotoxic agents. Their chemical structures were confirmed by spectral and elemental analysis; cytotoxic activity evaluation was performed against HEPG2, HCT116, and MCF7 cancer cell lines using the sulforhodamine-B assay. All the tested compounds except 6a showed high potency against the HEPG2 cancer cell line (IC50 8–101 nM/mL); 11 compounds out of 15 proved to be potent against HCT116 cells (IC50 3–49 nM/mL), also 11 of the tested compounds showed high potency against MCF7 cells with IC50 values ranging from 7 to 63 nM/mL. The rest of the tested compounds showed IC50 values of more than 100 nM/mL. Compounds 3e and 4d are the most active compounds against HEPG2 cells; in addition, 3e is the most active compound against MCF7 cells. Also, compounds 4a, 3a, and 3b are the most active compounds against HCT116 cells. Compounds 3a, 3b, 3e, 4a, and 4d were also evaluated for their inhibitory activity against the EGFR tyrosine kinase (EGFR-TK) and showed a percentage inhibitory activity ranging from 53 to 84%. The most potent EGFR-TK inhibitors, 3a (84%), 3b (75%), and 3e (60%), were docked into the ATP binding site of the EGFR to explore their binding mode and possible interactions. Keywords: Antitumor / Docking / EGFR-TKI / 2-Furyl / Quinazolinone / Tyrosine kinases Received: December 24, 2014; Revised: March 9, 2015; Accepted: March 20, 2015 DOI 10.1002/ardp.201400468
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Introduction Quinazoline is a heterocyclic system of two fused six-membered aromatic rings, benzene and pyrimidine. Quinazolines have drawn more and more attention in the field of medicinal chemistryduetotheirsignificantanddifferentbiologicalactivities as antidiabetes [1], anticancer [2], antibacterial [3], anti-inflammation [4], antivirus [5], antipsychotic [6], and antiobesity [7].
Quinazolin-4-one is a heterocyclic scaffold occupying a unique role in the field of medicinal chemistry and represents an important scaffold for designing new bioactive anticancer agents [8]. 2,3-Disubstituted quinazolin-4(3H)-one I (Fig. 1) was reported to have good anticancer activity [9], in addition, compound II [8] showed a promising antitumor activity. 2-(2Thieno)-6-iodo-3-amino-3,4-dihydro-quinazolin-4-one III [10] exhibited a remarkable anticancer activity in comparison to the known drug 5-FU. Addition of lipophilic moiety at position 2 as furan-2-yl to quinazolin-4-one scaffold afforded the active anticancer agent IV [11], the previously reported
Correspondence: Dr. Amany Belal, Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt. E-mail: [email protected] Fax: 0020822317958
*Additional correspondence: Dr. Marwa F. Ahmed, E-mail: [email protected]
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O
CH3 N
O
NH2
N
N H
N O
N
Cl
I
II
Cl
O
O
I
NH
N
N
N S
N
O
N
IV
III
Figure 1. Anticancer active quinazolin-4-one derivatives.
work has proved that changing the para position of 3-phenyl moiety has a great impact on anticancer activity [12], as a result we synthesized the quinazolin-4-one scaffold having a lipophilic furan moiety at position 2 with various substituents at the 4 position of the aryl moiety to study these structural changes in correlation to the activity. It also was of our interest to substitute the 4 position of the 3-phenyl with biologically active anticancer moieties as chalcone [13], pyrazole [14], isoxazole [15], and pyrimidine [16].
O
O O
N
H2N
O
COCH3
N
COCH3
O
N
f usion
It has been noticed that epidermal growth factor receptor (EGFR) is overexpressed in approximately 90% of tumors [17]. It is found to be the most important target molecules to date [18]. Quinazolines are well-known inhibitors for these receptors and are, therefore, expected to have a great therapeutic potential in cancer treatment [19, 20]. Moreover, lapatinib, gefitinib, erlotinib, and canertinib are quinazoline derivatives that have proved to act as EGFR tyrosine kinase inhibitors [21]. All these facts encouraged us to investigate
2
1
ArCHO NaOH O a, Ar =
d, Ar =
b, Ar =
OMe
c, Ar =
CH 3
Ar
O
Cl
N
HO e, Ar =
N
O
3a–e
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Scheme 1. Synthesis of compounds 3a–e.
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EGFR-TK inhibitory properties of the best active compounds of the newly designed quinazolines in this study. Furthermore, molecular docking studies were also performed to explore the binding mode and possible interactions with EGFR-TK active site.
Results and discussion Chemistry Compound 1 was synthesized according to the previously reported method [22]. Fusing compound 1 with p-aminoacetophenone at 160°C afforded 3-(4-acetylphenyl)-2(furan-2-yl)quinazolin-4(3H)-one 2 (Scheme 1), 1H NMR spectrum showed the appearance of additional aromatic protons and a singlet signal at 2.5 ppm indicating COCH3 protons. Claisen-Schmidt condensation of the acetyl derivative 2 with different aldehydes namely benzaldehyde, p-anisaldehyde, p-tolualdehyde, p-chlorobenzaldehyde, or 2-hydroxy benzaldehyde in ethanolic sodium hydroxide solution afforded the corresponding a,b-unsaturated ketones (chalcones) 3a–e, respectively (Scheme 1). Their 1H NMR spectra revealed the appearance of
additional aromatic protons and two duplets characterizing the two protons of chalcone moiety. Cyclocondensation of the unsaturated ketone 3a,b with hydrazine hydrate in absolute ethanol afforded the corresponding pyrazoline derivatives 4a,b. While cyclocondensation of the unsaturated ketone 3a,b with hydrazine hydrate in glacial acetic acid afforded the corresponding acetyl pyrazoline derivatives 4c,d (Scheme 2). 1H NMR spectra of 4a,b showed the appearance of CH2, CH, and NH signals indicating pyrazole moiety and that of compounds 4b,c showed signals for CH2, CH of pyrazole in addition to COCH3 signal. Moreover, their structures were also confirmed by mass, IR, 13 C NMR, and elemental analysis (Experimental section). Reacting a,b-unsaturated ketones 3a,b with hydroxylamine hydrochloride in ethanolic sodium hydroxide solution afforded the corresponding isoxazolines 5a,b, respectively, 1 H NMR revealed the appearance of CH2 and CH (isoxazoline protons). Cyclocondensation of the chalcones 3a,b with urea in the presence of HCl or with thiourea in the presence of NaOH afforded the corresponding tetrahydropyrimidinone 6a,b or tetrahydropyrimidine-thione derivatives 6c,d, respectively (Scheme 2). 1H NMR spectra of 6a–d revealed the appearance of CH2, CH, and NH signals characterizing the
O O
Ar N
O
N
thiourea
3a–e NH2NH2
NH2NH2
98%
EtOH
NH2OH
CH3COOH
N
O
Ar O
N
N Ar
N
O
H Ar
N
O
O
N
O 5a,b
4a–d a, Ar =
R=H
a, Ar =
b, Ar =
R=H
b, Ar =
OCH3
c, Ar = d, Ar =
X
N N
N
urea HCl
N O
R N
NaOH/EtOH
6a–d a, X = O, Ar = OCH3
R = COCH3 OCH3
R = COCH 3
b, X = O, Ar =
OCH3
c, X = S, Ar = d, X = S, Ar =
OCH3
Scheme 2. Synthesis of the target compounds 4a–d, 5a,b and 6a–d.
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tetrahydropyrimidine moiety, in addition IR, 13C NMR, mass, and elemental microanalysis were used as a tool in structures confirmation (Experimental section).
selective toward HCT116 and MCF7, respectively, than HEPG2 cell line.
SAR
Biological activity In vitro cytotoxic activity In the present work, the cytotoxic activity of the newly synthesized compounds was evaluated in vitro on liver (HEPG2), colon (HCT116), and breast (MCF7) cancer cell lines, using sulforhodamine-B (SRB) assay method and doxorubicin as a reference drug. Surviving fraction of cancer cells was plotted against drug concentration to obtain the survival curve. IC50 values (the dose causing 50% inhibition of viable cells) of the tested compounds were calculated and data are presented in Table 1 that shows the IC50 values for the tested compounds and doxorubicin in nM/mL. In general, the new compounds exhibited relevant cytotoxic activity, they showed to be active on HEPG2 except compound 6a, IC50 range of the active compounds is 8–101 nM/mL, and compounds 3e and 4d were the best active compounds with IC50 values equal to 8 and 9 nM/mL, respectively. The tested compounds, except 5a (its IC50 value exceeded 200 nM/mL), showed to be active on HCT116 cancer cell line with IC50 range from 3 to 150 nM/mL and compounds 3a, 3b, 3e, and 4a are the best active. Cytotoxic activity of the tested compounds against MCF7 was also promising as all of these compounds showed to be active, and compound 3e was the best active with IC50 value equal to 7 nM/mL. It is also noticed that some compounds showed cytotoxic selectivity toward specific cell lines, compound 3b showed to be 10 times more selective to HCT116 than HEPG2, compound 4a showed to be 20 times more selective on HCT116 than MCF7, finally compound 6a showed to be 100 and 50 times more
Substituting the para position of the 3-phenyl moiety with substituted chalcones afforded compounds 3a–e with an important cytotoxic activity on HEPG2, HCT116, and MCF7 cell lines, 4-chalcone moiety bearing 2-hydroxy phenyl was the best active and broad spectrum compound on the tested cancer cell lines, its IC50 ranges from 7 to 10 nM/mL. However, substituting the 4-chalcone moiety with 4-methylphenyl 3c or 4-chlorophenyl 3d leads to a decrease in activity on all the tested cell lines. Substituting the 3-phenyl moiety with chalcone bearing p-methoxyphenyl 3b or p-methylphenyl 3c leads to an increased activity and selectivity on colon cancer cell line HCT116. Substituting the para position of 3-phenyl moiety with pyrazole afforded compounds 4a–d with good cytotoxic activity, compound 4a has a 5-phenyl-1H-pyrazol-3-yl at the para position of 3-phenyl showed to be the best active on colon cancer cells, and compound 4d that has 1-acetyl-5-(4methoxyphenyl)-1H-pyrazol-3-yl at the 4 position of the 3-phenyl showed a highly potent activity on liver cancer cells (IC50 ¼ 9 nM/mL). Substituting the 3-phenyl with isoxazole moiety 5a,b leads to a decrease in activity; however, incorporating a 4-methoxyphenyl at the 5 position of isoxazol-3-yl moiety 5b gave a potent activity against HEPG2. Substituting the 3-phenyl with pyrimidine moiety 6a–d leads to good cytotoxic activity and the 2-thioxopyrimidines 6c,d were found to be more active than 2-oxopyrimidines 6a,b on HEPG2 and HCT116 cancer cell lines; however, the oxo derivatives 6a,b were found to be more active than the thioxo 6c,d on breast cancer cell line.
Table 1. In vitro anticancer activity of the newly synthesized quinazolines on liver (HEPG2), colon (HCT116), and breast (MCF7) cancer cell lines. IC50 (nM/mL SD) Comp. no. 3a 3b 3c 3d 3e 4a 4b 4c 4d 5a 5b 6a 6b 6c 6d Dox.
HEPG2 16.35 73.31 50.95 19.12 8.31 101.07 21.76 39.74 8.51 68.82 13.65 1781.13 50.74 166.6 19.09 5.66
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0.89 0.25 0.24 0.3 0.29 0.39 0.29 0.34 0.2 0.36 0.28 0.53 0.31 0.4 0.39 0.10
HCT116 7.67 6.63 49.24 38.04 9.73 2.86 122.18 113.74 22.51 223.78 150.4 11.04 40.38 10.24 15.47 4.79
0.97 0.18 0.19 0.15 0.23 0.43 0.49 0.44 0.37 0.35 0.3 0.29 0.21 0.37 0.36 0.45
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MCF7 42.42 123.27 111.2 15.71 6.49 55.31 43.38 29 30.70 115.75 114.22 24.81 15.76 29.62 62.98 4.44
0.63 0.24 0.23 0.13 0.28 0.37 0.38 0.23 0.3 0.26 0.42 0.41 0.39 0.41 0.36 0.11
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Table 2. EGFR tyrosine kinase assay of the best active quinazoline derivatives at 10 mM. Comp. no. 3a 3b 3e 4a 4d Erlotinib
% inhibition of EGFR tyrosine kinase 84 75 60 53 58 100
EGFR inhibition In this study, the best active compound on MCF7 (3e), the best active compounds on HEPG2 (3e and 4d), and the best active compounds on HCT116 (4a, 3a, and 3b) were screened at 10 mM for their inhibitory activity against EGFR-TK. The inhibitory range of the tested compounds is 53–84%, compound 3a is the most potent one, its inhibitory percent was at the value of 84%. Moreover, compounds 3a, 3b, and 3e that have chalcone substituted moiety attached to the para position of the 3-phenyl ring showed to be more potent inhibitors than derivatives bearing a pyrazole moiety 4a,d at the same position. The inhibitory percentage of chalcone
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substituted derivatives 3a, 3b, and 3e is 60–84%; however, the inhibitory percentage of pyrazole substituted derivatives 4a and 4d is 53–58% (Table 2). From the obtained data, we can say that the best active cytotoxic quinazolines 3a,b,e and 4a,d exhibited moderate to good activity against EGFR-TK. Compounds 3a and 3b, which showed a very good cytotoxic activity on HCT116 cell line, revealed inhibitory percentage against EGFR-TK equaling 84 and 75%, respectively; therefore, these two compounds can be considered as new leads for developing effective inhibitors against EGFR-TK. The other compounds 3e and 4a,d, which showed a good cytotoxic activity but had moderate (53–60%) inhibitory percentage against EGFR-TK, may need further exploration to detect their molecular targets.
Molecular docking studies The best active inhibitors of EGFR-TK, 3a, 3b, and 3e, were docked into the ATP binding site of EGFR to explore their binding mode and possible interactions with this receptor. All the calculations were performed using Molecular Operating Environment software (MOE) provided by the Chemical Computing Group, Canada, installed on Intel core i5, 2.5 G. EGFR cocrystallized with erlotinib (pdb: 1M17) [23] is the protein data bank file used in performing the docking studies. Docking was performed using London dG force and
Figure 2. 2D interactions of compound 3a with the ATP binding site of the EGFR.
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Figure 3. 2D interactions of compound 3b with the ATP binding site of the EGFR.
refinement of the results was done using Force field energy. Validation process was performed by docking the cocrystallized ligand (erlotinib) into ATP binding site of EGFR-TK to study the scoring energy (S), root mean standard deviation (rmsd), and amino acid interactions. Erlotinib is fitted into the active site pocket with S ¼ 21.66 kcal/mol and rsmd ¼ 1.4682, it also showed hydrogen bonding with Met 769 and another hydrogen bonding interaction with Thr 766 mediated by a molecule of water. The selected compounds were docked into ATP binding site of EGFR, they showed good fitting into the binding site with docking score energy equal to 20.93 (3a), 18.88 (3b), and 17.97 (3e). Moreover, compound 3a showed a hydrogen bonding with Cys 773 and Lys 721 amino acids through the CO of chalcone moiety and N-1 of quinazolinone nucleus, respectively, in addition to arene–arene interaction between the furyl moiety and Phe 699 amino acid (Fig. 2). Compound 3b showed two hydrogen bonds with Cys 773 and Thr 766 amino acids through CO of the quinazolinone and O of the methoxy group, respectively (Fig. 3). Compound 3e showed only one hydrogen bond with Asp 851 through H of the 2-hydroxyl group (Fig. 4). The 3D molecular surface map of the most
From this study, we can conclude that quinazolin-4-one bearing a 2-furyl moiety at position 2 and a phenyl moiety at position 3 proved to be an important scaffold for designing new cytotoxic active agents against HEPG2, HCT116, and MCF7 cell lines. Substituting the para position of 3-phenyl moiety in this scaffold with chalcone bearing 2-hydroxyphenyl revealed a broad spectrum anticancer agent 3e against the three cancer cell lines and substituting it with 4-methoxyphenyl 3b or 4-phenyl 3a afforded highly potent and selective compounds toward colon cancer cell line HCT116. However, 4-para chlorophenyl 3d and 4-methylphenyl 3c substituents lead to decrease in the activity. Substituting the para position of 3-phenyl ring with pyrazole leads to more active compounds than isoxazole and pyrimidine substituents. Compounds bearing a chalcone and pyrazole moiety at the para position of 3-phenyl ring 3a,b,e and 4a,d showed to act as good
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active compounds 3a, 3b, and 3e docking into the EGFR binding site is shown in Fig. 5.
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Figure 4. 2D interactions of compound 3e with the ATP binding site of the EGFR.
inhibitors for EGFR-TK. Chalcone derivatives 3a,b,e were found to be more potent inhibitors than pyrazole derivatives 4a,d. This work helped in finding potent inhibitors of EGFR belonging to new chemical sub-classes of quinazolinones (3a,b) and this opens the opportunity to expand further drug discovery projects. Molecular docking studies helped in understanding the various interactions between the ligands
and EGFR binding site in detail and this may help in design of novel potent EGFR-TK inhibitors.
Experimental Chemistry All melting points are uncorrected, elemental analyses were carried out in the microanalytical unit of National Research Centre and Cairo University, Egypt. IR spectra were recorded on FT-IR spectrophotometer Nexus 670Nicolet (USA) and Perkin Elmer-9712 spectrophotometer (Waltham, MA, USA). 1H NMR spectra were determined on a Varian-Gemini-300 MHz and Jeol-Ex 270 MHz NMR spectrometer using TMS as an internal standard. 13C NMR (DMSO-d6) spectra were recorded at 100.62 MHz at the aforementioned research center in Cairo University. Mass spectra were determined on Finnigan Mat SSQ 7000, mode EI 70 eV (Thermo Instrument Systems, Inc., USA). Thin layer chromatography was carried out on silica gel 60 F254 (Merck) plates using chloroform/petroleum ether/methanol (7:4:1) as an eluent system.
3-(4-Acetylphenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (2) Figure 5. 3D molecular surface map showing the docked poses of the most active compounds 3a (green), 3b (red), and 3e (blue) into EGFR binding site (PDB ID: 1M17).
A mixture of the benzoxazine 1 (2.13 g, 0.01 mol) and p-aminoacetophenone (1.35 g, 0.01 mol) was heated together upon fusion at 160°C on sand bath for 2 h. After cooling the crude mass was crystallized from ethanol twice to give dark
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brown crystals of compound 2, m.p. 250°C, 80% yield. Analysis calculated for C20H14N2O3 (330.34); calcd.: % C, 72.72; H, 4.27; N, 8.48, found: % C, 72.52; H, 4.17; N, 8.40. IR: ymax/cm1 3390 (C–H aromatic), 1700 (C –– O of acetyl), 1680 (C –– O of quinazolinone), 1633 (C –– N), and at 1590 (C –– C). 1H NMR (DMSO-d6, ppm): d 2.5 (3H, s, COCH3), 6.5–8.1 (m, 11H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 197, 164, 160.6, 144.5, 143, 141.7, 137.1, 136.6, 133.4, 129, 127.3, 124.3, 120.7, 109.9, 109.4, 26.6. MS (m/z, R.I.): calcd. for C20H14N2O3: 330.10; found: 330.
General method for the preparation of 3a–e A mixture of the ketone 2 (0.002 mol) and the appropriate aromatic aldehyde (0.002 mol) in ethanol (10 mL) was prepared. 5% NaOH in ethyl alcohol (10 mL) was added dropwise within 15 min. The reaction mixture was refluxed for 3 h then cooled and the crude precipitated material was filtered off, air dried, and then crystallized from the proper solvent to give the chalcones 3a–e, respectively.
(E)-3-(4-Cinnamoylphenyl)-2-(furan-2-yl)quinazolin-4(3H)one (3a) Crystallized from ethanol to give yellow crystals, m.p. 155°C in 70% yield. Analysis for C27H18 N2O3 (418.44), calcd. %C, 77.50; H, 4.34; N, 6.69; found: %C, 77.45; H, 4.30; N, 6.62; IR: ymax/cm1 1690 (C –– O quinazolinone), 1665 (C –– O of the a,bunsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSO-d6, ppm): 6.6–6.8 (2H, d, d, CH –– CH) and 6.9–7.9 (m, 16H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 189.7, 165, 160.6, 145.2, 144.5, 142.7, 140.5, 139.5, 135.2, 134.5, 134.2, 131.4, 129.7, 129.5, 127.9, 127.2, 125.9, 125.4, 124.9, 121.3, 120.6, 109.9, 109.4. MS (m/z, R.I.): calcd. for C27H18N2O3: 418.13; found: 418.13.
(E)-2-(Furan-2-yl)-3-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)quinazolin-4(3H)-one (3b) Crystallized from glacial acetic acid to give yellow crystals, m.p. 190°C in 75% yield. Analysis for C28H20N2O4 (448.47), calcd. % C, 74.99; H, 4.50; N, 6.25; found: %C, 74.97; H, 4.49; N, 6.24; IR: ymax/cm1 1700 (C –– O quinazolinone), 1670 (C –– O of the a,bunsaturated ketone), and at 1600 (C –– N). 1H NMR spectrum (DMSO-d6, ppm): 3.80 (3H, s, OCH3), 6.8–6.9 (2H, d, d, CH –– CH) and 6.9–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 190.4, 166.7, 162.4, 159.9, 146.2, 145.9, 143.4, 141.7, 140.5, 133.5, 133.2, 131.4, 130.7, 127.7, 127.5, 126.6, 124.9, 124.3, 120.9, 114.2, 112.7, 112.4, 59.7. MS (m/z, R.I.): calcd. for C28H20N2O4: 448.14; found: 448.14.
(E)-2-(Furan-2-yl)-3-(4-(3-p-tolylacryloyl)phenyl)quinazolin-4(3H)-one (3c) Crystallized from glacial acetic acid to give yellow crystals, m.p. 240°C in 70% yield. Analysis for C28H20 N2O3 (432.47), calcd. %C, 77.76; H, 4.66; N, 6.48; found: %C, 77.72; H, 4.64; N, 6.46; IR: ymax/cm1 1700 (C –– O quinazolinone), 1665 (C –– O of the a,bunsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSO-d6, ppm): 2.5 (3H, s, CH3), 6.7–6.9 (2H, d, d, CH –– CH) and 6. 8–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 188.5, 165.3,
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161.2, 145.1, 144.9, 143.2, 140.6, 137.6, 134.5, 134.2, 132.2, 131.9, 128.9, 128.5, 127.3, 126.7, 125.7, 122.3, 120. 8, 111.5, 111, 22.3. MS (m/z, R.I.): calcd. for C28H20N2O3: 432.15; found: 432.15.
(E)-3-(4-(3-(4-Chlorophenyl)acryloyl)phenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (3d) Crystallized from glacial acetic acid to give yellow crystals, m.p. 250°C in 70% yield. Analysis for C27H17ClN2O3 (452.89), calcd. %C, 71.60; H, 3.78; N, 6.19, found: %C,71.59; H, 3.69; N, 6.17. IR: ymax/cm1 1710 (C –– O quinazolinone), 1670 (C –– O of the a,b-unsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSOd6, ppm): 6.7–6.9 (2H, d, d, CH –– CH) and 6. 8–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 190.2, 165.7, 161.9, 146.1, 143.9, 141.7, 138.5, 135.6, 135.5, 135.2, 131.4, 129, 128.7, 127.9, 125.6, 125.2, 124.9, 122.3, 120.9, 109.5, 109. MS (m/z, R.I.): calcd. for C27H17ClN2O3: 452.09; found: 452.09.
(E)-2-(Furan-2-yl)-3-(4-(3-(2-hydroxyphenyl)acryloyl)phenyl)quinazolin-4(3H)-one (3e) Crystallized from ethanol to give brown crystals, m.p. 159°C in 70% yield. Analysis for C27H18N2O4 (434.44), calcd. %C, 74.64; H, 4.18; N, 6.45; found: %C, 74.60; H, 4.11; N, 6.43; IR: ymax/cm1 3380 (OH), 1720 (C –– O quinazolinone), 1675 (C –– O of the a,b-unsaturated ketone) and at 1600 (C –– N). 1H NMR (DMSO-d6, ppm): 6.7–6.9 (2H, d, d, CH –– CH), 6. 8–8.2 (m, 15H, Ar-H, and furan-H) and 11.5 (s, 1H, O–H). 13C NMR (DMSO-d6): 188.9, 166.7, 160.7, 157.1, 145.2, 143, 142.4, 141, 138.9, 133.5, 133.3, 131.4, 129.3, 128.9, 127.3, 126.7, 126.5, 124.9, 122.6, 121.9, 121.3, 120.7, 117.6, 109.7, 109.2. MS (m/z, R.I.): calcd. for C27H18N2O4: 434.13; found: 434.13.
General method for the preparation of 4a,b A mixture of the chalcone 3a,b (0.005 mol) and hydrazine hydrate (2.5 mL, 0.005 mol, 98%) in abs. ethanol (25 mL) was heated under reflux for 10 h. After cooling, the separated material was filtered off, air dried, and then crystallized from the proper solvent to give 4a,b, respectively.
2-(Furan-2-yl)-3-(4-(5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)quinazolin-4(3H)-one (4a) Crystallized from ethanol to give yellow crystals, m.p. 200°C in 70% yield. Analysis for C27H20N4O2 (432.47), calcd. %C, 74.92; H, 4.54; N, 12.90, found: %C, 74.98; H, 4.66; N, 12.95; IR: ymax/cm1 3385 (NH), 3030 (CH, aromatic), 1670 (C –– O), and 1635 (C –– N). (DMSO-d6, d ppm): 3.3 (d,d, 2H, CH2, pyrazoline ring, J ¼ 5.6 Hz), 3.90 (t, 1H, CH of pyrazoline, 7.21 (s, NH, exchangeable with D2O), 6. 8–8.2 (m, 16H, Ar-H, and furan-H). 13 C NMR (DMSO-d6) d ppm: 164.3, 161.7, 151.7, 144.5, 143.5, 141.7, 135.9, 133.4, 132, 129.4, 128.5, 127.3, 126.9, 126.7, 126.6, 124.5, 120.6, 121.9, 110.5, 110.2, 49.1, 42.6. MS (m/z, R.I.): calcd. for C27H20N4O2: 432.16; found: 432.16.
2-(Furan-2-yl)-3-(4-(5-(4-methoxyphenyl)-4,5-dihydro-1Hpyrazol-3-yl)phenyl)quinazolin-4(3H)-one (4b) Crystallized from ethanol to give brown crystals, m.p. 220°C in 70% yield. Analysis for C28H22N4O3 (462.50), calcd. %C,
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72.71; H, 4.79; N, 12.11; found: %C, 72.65; H, 4.75; N, 12.00; IR: ymax/cm1 3390 (NH), 3035 (CH, aromatic), 1670 (C –– O), and 1640 (C –– N). 1H NMR (DMSO-d6, ppm) 3.4 (d,d, 2H, CH2, pyrazoline ring, J ¼ 5.6 Hz), 3.70 (3H, s, OCH3), 3.90 (t, 1H, CH of pyrazoline), 7.4 (s, NH, exchangeable with D2O), 6.9–8.3 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 166.2, 162.4, 158.6, 152.6, 145.7, 144.2, 143.7, 135.8, 135.4, 133.4, 132, 130.2, 127.3, 126.7, 126.5, 125.4, 120.8, 114.1, 109.9, 109.2, 55.8, 50.2, 43.9. MS (m/z, R.I.): calcd. for C28H22N4O3: 462.17; found: 462.17.
General method for the preparation of 4c,d A mixture of the chalcone 3a,b (0.005 mol) and hydrazine hydrate (2.5 mL, 0.005 mol, 98%) in the presence of (10 mL) glacial acetic acid was heated under reflux for 10 h. After cooling, the mixture was poured into ice/water and filtered off, air dried, and then crystallized from the proper solvent to give 4c,d, respectively.
3-(4-(1-Acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (4c) Crystallized from ethanol to give dark brown crystals, m.p. 192°C in 70% yield. Analysis for C29H22N4O3 (474.51), calcd. % C, 73.40; H, 4.67; N, 11.81; found: %C, 73.35; H, 4.64; N, 11.80; IR: ymax/cm1 3030 (CH, aromatic), 1690 (C –– O quinazolinone, 1670 (C –– O, acetyl), and 1635 (C –– N). 1H NMR (DMSO-d6, ppm) 2.1 (3H, s, COCH3), 3.3 (d, d, 2H, CH2, pyrazoline ring), 3.90 (t, 1H, CH of pyrazoline, 6.8–8.2 (m, 16H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 168.5, 164.9, 160.6, 151.6, 144.3, 143.4, 141.9, 135.2, 135.9, 129.2, 128.5, 127.6, 126.9, 126.6, 126.4, 124.4, 123.7, 112.7, 112.2, 58.9, 39.3, 23.4. MS (m/z, R.I.): calcd. for C29H22N4O3: 474.17; found: 474.17.
3-(4-(1-Acetyl-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol3-yl)phenyl)-2-(furan-2-yl)quinazolin-4(3H)-one (4d) Crystallized from ethanol to give brown crystals, m.p. 149°C in 65% yield. Analysis for C30H24N4O4 (504.54), calcd. %C, 71.42; H, 4.79; N, 11.10; found: %C, 71.40; H, 4.75; N, 11.95. IR: ymax/cm1 3035 (CH, aromatic), 1695 (C –– O quinazolinone, 1670 (C –– O, acetyl), and 1640 (C –– N). 1H NMR (DMSO-d6, ppm) 2.2 (3H, s, COCH3), 3.4 (d, d, 2H, CH2, pyrazoline ring), 3.70 (3H, s, OCH3), 3.95 (t, 1H, CH of pyrazoline), 6.9–8.3 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6): 169.4, 165.9, 162.6, 158.6, 151.4, 144.5, 143, 140.4, 136, 134, 133.2, 132, 130.2, 127.6, 127.3, 126.6, 124.5, 121.7, 114.1, 109.2, 109, 58.9, 56.9, 39.3, 23.4. MS (m/z, R.I.): calcd. for C30H24N4O4: 504.18; found: 504.18.
General method for the preparation of 5a,b A mixture of chalcone 3a,b (3 mmol) and hydroxylamine hydrochloride (5 mmol) in sodium hydroxide solution (0.5 g NaOH in 0.5 mL water) in ethanol (60 mL) was refluxed for 3 h. The product obtained upon cooling was filtered off, washed with water, and recrystallized from the proper solvents to obtain the desired compounds 5a,b, respectively.
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2-(Furan-2-yl)-3-(4-(5-phenyl-4,5-dihydroisoxazol-3-yl)phenyl)quinazolin-4(3H)-one (5a) Crystallized from ethanol to give brown crystals, m.p. 235°C in 65% yield. Analysis for C27H19N3O3 (433.46), calcd. %C, 74.81; H, 4.42; N, 9.69; found: %C, 74.79; H, 4.40; N, 9.64. IR: ymax/cm1 3030 (CH, aromatic), 1665 (C –– O), and 1630 (C –– N). 1 H NMR (DMSO-d6, d ppm): 3.6–3.72 (d, d, 2H, CH2, isoxazoline ring), 6.24 (t, 1H, CH, isoxazoline ring), 6.5–8.1 (m, 16H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 164.4, 160.9, 156.2, 143.9, 143.3, 142.4, 141.6, 135, 133.5, 130.5, 129.4, 130.2, 128.6, 128.3, 128.1, 126.7, 126.5, 126, 124.7, 120.8, 112.7, 112.4, 82.8, 42. MS (m/z, R.I.): calcd. for C27H19N3O3: 433.14; found: 433.14.
2-(Furan-2-yl)-3-(4-(5-(4-methoxyphenyl)-4,5dihydroisoxazol-3-yl)phenyl)quinazolin-4(3H)-one (5b) Crystallized from glacial acetic acid to give brown crystals, m.p. 155°C in 65% yield. Analysis for C28H21N3O4 (463.48), calcd. % C, 72.56; H, 4.57; N, 9.07; found: %C, 72.51; H, 4.52; N, 9.03. IR: ymax/cm1 3040 (CH, aromatic), 1670 (C –– O), and 1630 (C –– N). 1 H NMR (DMSO-d6, d ppm): 3.7–3.8 (d, d, 2H, CH2, isoxazoline ring), 3.9 (3H, s, OCH3), 6.24 (t, 1H, CH, isoxazoline ring and 6.7–8.2 (m, 15H, Ar-H, and furan-H). 13C NMR (DMSO-d6) d ppm: 165, 160.4, 159.4, 156.6, 144.9, 143.2, 142.4, 134.7, 133.4, 132, 129.6, 127.3, 127, 125.9, 125.7, 125, 124.5 120.8, 114.5, 110.7, 109.2, 82.8, 55.8, 42. MS (m/z, R.I.): calcd. for C28H21N3O4: 463.15; found 463.15.
General method for the preparation of 6a,b A mixture of the chalcone 3a,b (0.005 mol) and urea (0.5 g, 0.005 mol) in ethanol (20 mL) and conc. HCl (5 mL) was refluxed for 7 h. The reaction mixture was concentrated to half its volume, cooled, and neutralized with NH4OH solution. The precipitated solid was filtered off, washed with water, air dried, and crystallized from the proper solvent to give compound 6a,b.
2-(Furan-2-yl)-3-(4-(2-oxo-6-phenyl-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6a) Crystallized from glacial acetic acid to give brown crystals, m.p. 205°C in 60% yield. Analysis for C28H20N4O3 (460.48), calcd. % C, 73.03; H, 4.38; N, 12.17; found: %C, 73.00; H, 4.34; N, 12.11. IR: ymax/cm1 3215–3600 (OH enolic of pyrimidine), 3040 (CH, aromatic), 1690 (C –– O), and 1630 (C –– N). 1H NMR (DMSO-d6, d ppm): 3.4 (2H, d, CH2 of pyrimidinone), 5.6 (1H, t, CH of pyrimidinone), and 6.4–8.1 (m, 17H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 164.6, 164, 163, 160.6, 144.5, 143.5, 143, 141.7, 136.2, 133.5, 129.4, 128.5, 127.3, 126.9, 126.7, 126.6, 124.5 121.2, 109.9, 109.4, 43.6, 42.7. MS (m/z, R.I.): calcd. for C28H20N4O3: 460.15; found: 460.15.
2-(Furan-2-yl)-3-(4-(6-(4-methoxyphenyl)-2-oxo-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6b) Crystallized from ethanol to give brown crystals, m.p. 150°C in 60% yield. Analysis for C29H22N4O4 (490.51), calcd. %C, 71.01; H, 4.52; N, 11.42; found: %C, 71.94; H, 4.49; N, 11.40; IR:
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Arch. Pharm. Chem. Life Sci. 2015, 348, 487–497 M. F. Ahmed and A. Belal
ymax/cm1 3220–3610 (OH enolic of pyrimidine), 3055 CH, aromatic), 1690 (C –– O), and 1635 (C –– N). 1H NMR (DMSO-d6, d ppm): 3.5 (2H, d, CH2 of pyrimidinone), 3.9 (3H, s, OCH3), 5.7 (1H, t, CH of pyrimidinone), and 6.4–8.4 (m, 16H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 166.2, 165, 164.5, 162.3, 158.6, 145.2, 143.3, 142.9, 137.5, 135.8, 135, 133.4, 129.9, 127.3, 126.7, 126.6, 125.2, 120.8, 114.1, 113.3, 113, 56.8, 43.9, 40.5. MS (m/z, R.I.): calcd. for C29H22N4O4: 490.16; found: 490.16.
General method for the preparation of 6c,d A mixture of the chalcone 3a,b (0.005 mol) and thiourea (0.005 mol) in the presence of 0.5 g of NaOH in 5 mL of water. The mixture was refluxed in (25 mL) of ethanol for 6 h then concentrated under vacuum and neutralized with diluted HCl. The precipitated material was filtered off, washed with water, dried, and crystallized from ethanol to give 6c,d.
2-(Furan-2-yl)-3-(4-(6-phenyl-2-thioxo-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6c) Crystallized from ethanol to give brown crystals, m.p. 270°C in 60% yield. Analysis for C28H20N4O2S (476.55), calcd. %C, 70.57; H, 4.23; N, 11.76; found: %C, 70.51; H, 4.20; N, 11.73; IR: ymax/cm1 3210–3590 (OH enolic of pyrimidine), 3030 (CH, aromatic), 1690 (C –– O), 1630 (C –– N), and 1270 (C –– S). 1H NMR (DMSO-d6, d ppm): at 3.5 (2H, d, CH2 of pyrimidinone), 5.7 (1H, t, CH of pyrimidinone), and 6.5–8.1 (m, 17H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 190.2, 164.5, 165, 160.4, 158.9, 146.7, 145, 143, 143.4, 139.2, 137.6, 135.7, 133.6, 130.5, 129.4, 128.7, 128.5, 126.5, 123.9, 114.9, 114.2, 57.8, 53.7, 43.5. MS (m/z, R.I.): calcd. for C28H20N4O2S: 476.13; found: 476.13.
2-(Furan-2-yl)-3-(4-(6-(4-methoxyphenyl)-2-thioxo-1,2,5,6tetrahydropyrimidin-4-yl)phenyl)quinazolin-4(3H)-one (6d) Crystallized from ethanol to give brown crystals, m.p. 215°C in 60% yield. Analysis for C29H22N4O3S (506.57), calcd. %C, 68.76; H, 4.38; N, 11.06; found: %C, 68.71; H, 4.35; N, 11.04; IR: ymax/cm1 3215–3590 (OH enolic of pyrimidine), 3035 (CH, aromatic), 1690 (C –– O), 1630 (C –– N), and 1275 (C –– S). 1H NMR (DMSO-d6, d ppm): at 3.5 (2H, d, CH2 of pyrimidinone), 3.8 (3H, s, OCH3), 5.7 (1H, t, CH of pyrimidinone), 6.5–8.1 (m, 16H, Ar-H, pyrimidone, and furan-H). 13C NMR (DMSO-d6): 189, 167.4, 166.1, 161.6, 144.2, 143.7, 143, 141.9, 136.2, 135, 132.7, 129.4, 128.4, 127.9, 126.9, 126.7, 124.5, 122.9, 111.9, 111.2, 52, 42.9. MS (m/z, R.I.): calcd. for C29H22N4O3S: 506.14; found: 506.14.
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University, Cairo, Egypt). Reagents and chemicals were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). Cells were seeded for 24 h in a 96-well microtiter plates at a concentration of 1000–2000 cells/well, 100 mL/well, then cells were incubated for 48 h with various concentrations (0, 6.25, 12.5, 25, 50, and 100 mg/mL) of the tested compounds and doxorubicin, three wells were used for each concentration; after incubation for 48 h, the cells were fixed with 10% trichloroacetic acid 150 mL/well for 1 h at 4°C, washed by distilled water for three times. Wells were stained for 10–30 min at room temperature with 0.4% SRB, dissolved in 1% acetic acid 70 mL/well. Washed with acetic acid 1% to remove unbound dye till colorless drainage obtained. The plates were subjected to air drying, 24 h not exposed to UV. The dye was solubilized with 150 mL/well of 10 mM Tris-EDTA (pH 7.4) for 5 min on a shaker at 1600 rpm. The optical density of each well was measured spectrophotometrically at 545 nm with an ELISA microplate reader. The percentage of surviving cells was calculated and plotted against different concentrations of the tested compounds to obtain the survival curve. The IC50 values were calculated using sigmoidal concentration–response curve fitting models (Sigmaplot software).
EGFR TK inhibition assay Compounds 3a, 3b, 3e, 4a, and 4d were tested in vitro for inhibition of EGFR tyrosine kinase using Kinase-Glo Plus luminescence kinase assay kit. The tested compounds were dissolved in DMSO. They were then added to reaction plates containing the EGFR tyrosine kinase in assay buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 10 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 0.02% Brij35, 0.02 mg/mL bovine serum albumin, 0.1 mM Na3VO4, 2 mM dithiothreitol, 1% DMSO). Reactions were initiated by addition of a mixture of ATP (Sigma–Aldrich) and 33P ATP (Perkin Elmer) to a final concentration of 10 mM. Reactions were carried out at room temperature for 2 h, followed by spotting of the reactions onto P81 ion exchange filter paper (Whatman, Inc., Piscataway, NJ, USA). Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid [25]. The results are presented as percentage enzyme inhibition in comparison to erlotinib as a reference EGFR-TK inhibitor.
Molecular docking
Cytotoxic activity of the newly synthesized compounds was evaluated using the SRB assay [24]. HEPG2, HCT116, and MCF7 cancer cell lines were obtained from the American Type Culture Collection (ATCC, MN, USA) through the Tissue Culture Unit (The Egyptian Organization for Biological Products and Vaccines, Vacsera, Egypt). Cytotoxicity was performed at the Center for Genetic Engineering (Al-Azhar
Molecular modeling studies were carried out on an Intel Core i5, 2.53 GHz processor, 4 GB RAM memory with Windows XP 32-bit operating system using Molecular Operating Environment software. Energy minimization was performed with MOE, RMSD gradient of 0.05 kcal/mol, MMFF94X Force field and the partial charges were automatically calculated. The X-ray crystallographic structure of erlotinib cocrystallized with EGFR was obtained from the protein data bank (PDB ID: 1M17). The ATP binding site of EGFR was prepared for docking studies where erlotinib was removed from the active site; hydrogen atoms were added to the structure with their standard geometry; MOE Alpha Site Finder was used for the
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Pharmacological screening In vitro cytotoxicity
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active sites detection; the obtained model was saved as moe file then used in predicting interactions at the active site between the selected compounds and EGFR; and the 2D structures of the docked compounds were generated by ChemDraw, transformed to their 3D by Moe program, protonated, and subjected to energy minimization, then saved as mdb file to be docked into the active site of EGFR. The authors have declared no conflict of interest.
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