Molecular modeling study bioactive natural product of khellin analogues as a novel potential pharmacophore of EGFR inhibitors

Abstract

Khelline is naturally occurring furochromone exhibited significant Epidermal Growth Factor Receptor (EGFR) inhibitory activity. The newly synthesized compounds 2–5 displayed the most potent EGFR inhibitory activity on MCF-7 and HeLa. In vitro study against 59 different human tumour cell lines derived from nine cancer type in NCI (USA), which was presented and documented. Molecular docking simulation was performed to position compounds 1–5 into the EGFR active site to determine the probable binding mode.

Keywords::

• EGFR inhibitor
• anticancer
• furochromone
• khellin
• molecular docking

Introduction

The chemotherapy of cancer has entered a new era of molecular targeted therapeutics, which was highly selective and not associated with the serious toxicities of conventional cytotoxic drugs¹. Receptor protein tyrosine kinases play an important role in signal transduction pathways that regulate cell division and differentiation. Among the growth factor receptor kinases that have been identified as being important in cancer chemotherapy. Activation of EGFR may be because of overexpression, mutations resulting in constitutive activation, or Autocrine expression and ontogenetic functions of the PACAP ligand/receptor system during sympathetic development^2–5. The role of EGFR has been most thoroughly studied in breast cancer, where it is overexpressed in 25–30% of cases and is correlated with a poor prognosis. EGFR overexpression is also seen in ovarian cancer⁶, lung cancer (especially lung adenocarcinomas)^7–9 and in hormone-refractory prostate cancer¹⁰. Compounds that inhibit the kinase activity of EGFR after binding of its cognate ligand are of potential interest as new therapeutic antitumor agents^11,12. The mercapto of furochromone derivatives play important role in anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumorigenesis^13–15. For example, pyrazolopyrimidine urea bound to the ATP binding site of KDR kinase, the amino thienopyrimidine core mimics the adenine of ATP in its interaction with the hinge region of KDR. Hydrogen bonds are formed between the exocyclic amino group of pyrimidine and the backbone carbonyl of Glu 917 and the proximal ring nitrogen and the backbone N–H of Cys 919¹⁶. Many chromone-pyrimidine derivatives are acknowledged to possess a wide range of bioactivities. The furochromone-pyridines 4 and 5 motif makes up the core structure of numerous biologically active compounds.

To the best of our knowledge, few reports have been dedicated to the synthesis and EGFR inhibitory activity of furochromones containing thieno skeleton^17,18. The present work deal with the synthesis and structure activity relationships (SAR) of a series of furochromone 2, 3, 4, derivatives containing thieno skeleton as anticancer agents. It was observed that compounds 3, 4 and 5 displayed the most potent EGFR inhibitory activity with IC₅₀ of 0.12 into 0.04 µM, which was comparable to the positive control erlotinib (IC₅₀ = 0.03 µM). Docking simulations were performed using the 3D structure of the EGFR in complex with inhibitor to explore the binding modes of these compounds at the active site.

In particular, benzofuran ring is a common moiety in many biologically active natural and therapeutic products^19–21, some of which having substituents, and represent a very important heterocyclic pharmacophore²². Khellin 1 was one of several furochromones that can be isolated from fruits and seeds of Ammi visnaga L., a perennial herbaceous plant that grows wild in many Eastern Mediterranean countries which possesses a high active constituent of many modern medicines of antitumor properties^23–34. The powder fruit of khella are used in Egyptian folk medicine as diuretics for treatment of kidney, bladder stones³⁵. The clinical and therapeutic effectiveness of khellin, a constituent of khella, with respect to coronary, respiratory and urologic indications, has been demonstrated in experiments. Many naturally occurring as well as synthetic compounds containing the pyridine scaffold exhibit interesting pharmacological properties³⁶. Enzymes like isomerase II^37,38 (topo II α) and thymidylate synthase³⁹ (TS) are the common targets of pyrimidine based anticancer drugs^40–42 are in clinical use. Heterocyclic like pyrimidine, pyridine is an integral part of a huge number of natural and synthetic compounds which play important roles in the biological systems^43–45.

Molecular docking used to understand drug–receptor interaction in modern drug design. We described here the docking of khellin as inhibitor to human activities against Epidermal Growth Factor Receptor (EGFR)⁶, on MCF-7 and HELA in comparison with erlotinib^7,48 as a reference drug. We docked EGFR derivates and study their specific interactions⁹, the result indicates that the molecular docking approach is reliable and produces a good correlation of five compounds between docking score and inhibitory activity IC₅₀ values. The docked complexes provide better insights to design more EGFR inhibitors prior to their synthesis⁵⁰.

Results and discussion

Chemistry

The synthesis of compounds 2, 3, 4 and 5 followed the general pathway and presented by study the reactivity of khellin 1 against sulphur (S₈) in the presence of malononitril CH₂(CN)₂ under basic condition to provide 2-amino-4,8-dimethoxy-10-methyl-furo[2,3-g]thieno[2,3-c]chromene-3-carbonitriles 2 in low yield (0.25 g, 20% ). Three traits of action of compound 2 were simultaneously examined in a cross of P₂S₅, CS₂ and Ac₂O in refluxing pyridine to provide compound 3, 4 and 5 in moderate yield (60%, 50%, and 58% respectively). So that when compound 2 reacted with phosphorus pentasulphide [P₂S₅] gave 3 in (0.80 g, 60%), and with carbon disulphide [CS₂] gave 4 in (0.85 g, 50%). But with acetic anhydride gave thieno pyrimidinone derivatives 5, in (0.90 g, 58%) (Scheme 1).

Scheme 1.  Reaction of khellin (1) with malononitril gave compound (2) which was examined against P2S5, CS2 and Ac2O in refluxing pyridine.

In the proposed reaction, mechanism proposed in Scheme 2 described the formation of organophosphorus (OP) ring that feature of furochromone skeleton by the reaction of 2 with P₂S₅ gave phosphonites intermediate of pyridine complex V, which neutralized by addition of HCl to provide organophosphorus compound (OPC) of 2,3-dihydro-2-mercapto-2,1,3-phosphadiazine-4(1H)-thione-2-sulphide 3. Many of the most important biochemicals are organophosphates, including DNA and RNA as well as many cofactors that are essential for life⁵¹.

Scheme 2.  The Reaction Mechanism proposed for the formation of organophosphorus compound (OPC).

The compound has now synthesized in good yield by refluxing phosphorus pentasulphide in pyridine and it gives the expected analytical data for all the elements present and the presence of phosphorus was confirmed by ³¹P-NMR. Treatment of the complex V with cold aqueous alkali removal of the pyridine in ether, basification with sodium hydroxide (200 mL) and just acidifying with aqueous hydrochloric acid to precipitate the compound 3. In fact, in the IR spectra of the product V obtained (isolated as a pyridine solvate), there was no absorption band from the CN group. In the ³¹P NMR (121 MHz, DMSO-d6) of compound V, δ 11.96 observed the following proton signals at δ 2.30 (s, 3H, CH₃), 4.10 (s, 6H, 2CH₃), 4.25 (s, 1H, CH), 6.04 (s, 1H, CH), 6.96 (s, 1H, CH), 7.26-7.27 (d, 2H, CH), 7.83-7.87 (m, 1H, CH), 8.08 (s, 1H, CH), 8.11-8.12 (d, 1H, CH), 8.35 (s, 1H, NH), 8.80-8.82 (m, 1H, NH) and taking into account the results of previous work of pyridine protons at 7.79 (2H, t, β-H); 8.26 (1H, t, γ-H); 8.79 (2H, d, α-H). An SH proton signal is not clearly seen in the ¹H NMR data, which is consistent with the data in concerning a study of the spectra for such compounds⁵². The tendency of 1,3,2-diazaphosphorin derivatives to form pyridine solvates of variable composition has been described^53–57. We carried out an analogous cyclization within this work based on 1,2-trimethylene-4-amino-5-cyanoimidazole^58,59. Nevertheless, a stable compound was obtained upon heating pyridine solvate V with dilute HCl. The high basicity of the latter makes it possible to synthesize a stable compound 3, for which we obtained not only clear spectral data but also satisfactory elemental analysis results (see Experimental for more details).

According to the ¹HNMR data of 3–5, which exhibit only one set of signals, the reaction is highly stereoselective and results in the formation of only one diastereomers with the cis-arrangement of the substituents in the methyl tetrahydropyran ring. This is most likely related to the formation of an intramolecular hydrogen bond (the IR spectra of 3–5 in CHCl₃ solution had a bonded peak at 3470–3495 cm⁻¹, which were unchanged upon dilution). It seems probable that little, if any, OH···OC intramolecular hydrogen bonding interactions can occur in trans-isomer. Thus, as expected, the replacement of the amino cyano group in khellin by the pyrimidine ring and diazaphosphorine ring substantially increases the reactivity of the khellin system toward nucleophiles and makes it possible to design from its new organo phosphorus containing heterocycles, which are of interest as biologically active compounds. The data of these synthesized compounds was submitted to National Cancer Institute (NCI) for anti proliferative activity. As per the protocol of NCI, only three representative compounds of the series were selected and granted NSC codes viz; compound 2 (NSC751491), compound 4 (NSC751492), compound 5 (NSC751493), and screened at National Cancer Institute (NCI), USA for anti proliferative activity at a single high dose (10⁵ M) in full NCI 59 cell panel. Compound 5 (NSC 751493) exhibited the best result at single dose. This paper presents in vitro anticancer profile of all the selected compounds including the most potent compound of the against NCI’s 60 human cancer cell lines.

EGFR inhibitory activity and SAR of the khellin derivatives 1–5

Furochromone derivatives 1–5 were evaluated for their ability to inhibit the autophosphorylation of EGFR kinases using a solid-phase ELISA assay. The inhibition constant IC₅₀ of the compounds were summarized in Figure 2 and observed that khelline containing thienyl skeleton 2–5 have found to show fairly good inhibitory activity displaying IC₅₀ values between 0.15 to 0.04 µM. A series of furo-[2,3-g]thieno[2,3-c]chromene derivatives 2–5 have synthesized and tested against their EGFR kinase inhibitory activity. As illustrated in Figure 1, compounds 2–5 displayed moderate EGFR inhibitory activity with IC₅₀ ranging from 0.04 to 0.15 µM. These results indicated that the thienyl skeleton in the furo-[2,3-g]thieno[2,3-c]chromene derivatives 2, 4, 5 play an important role in the EGFR inhibitory activity. For example, there is only one difference between compound 1 and compounds 2–5, that is, the former four compounds have the thienyl skeleton. Compounds 2–5 exhibited potent EGFR inhibitory activity with IC₅₀ ranging from 0.04 to 0.15 µM, however, compound 3 showed moderate EGFR inhibitory activity with IC₅₀ more than that 0.12 µM. The same rule also has applied to compounds 1–5 in different cell line, see Figure 1. Subsequently, structure activity relationships (SAR) study was performed by modification of the parent compound to determine how the substituents of subunits affected the EGFR inhibitory activities. Inspection to the chemical structure of compounds 1–5, as shown in Figure 1 and Scheme 2, this led to it could be divided into two sub-units: I-, II-, III-, IV and V rings. The structure activity relationships (SAR) in compounds 1–5 having two methoxy group at 6-position 10-position gave a good EGFR inhibitor activity, as outlined in Figure 1. The most significantly of the stronger electron-donating groups at specific position and the more potent was attributed to the potency order OCH₃ > SH >OH > CH₃. Among them, compound 5 displayed the most potent EGFR inhibitor activity with IC₅₀ = 0.04 µM, which was comparable to the positive control erlotinib (IC₅₀ = 0.03 µM). Therefore, compound 5 with potent inhibitory activity in tumour growth inhibition would be a potential anticancer agent.

Figure 1.  The SAR and EGFR inhibitory activity of the khellin derivatives 1–5.

Figure 2.  The docking score of the tested compounds 1–5.

Antiproliferative activity and molecular docking study of the compounds 1–5

The binding affinity was evaluated by the hydrogen bonding and binding free energies (DGb, kcal/mol). Due to the most potent EGFR inhibitory activity of 5 was selected for further molecular docking study. Then, molecular docking of 5 into ATP binding site of EGFR kinase was performed on the binding model based on the EGFR complex structure (1M17.pdb). The binding model of compound 5 and EGFR are depicted in Figure 2. In the binding model, compound 5 is nicely bound to EGFR kinase with NH group toward the side chain of carbonyl group of D831 (Asp831), forming a more optimal H-bond interaction (distance: 1.94 Å, angle: 127.1°). Based on the significant EGFR inhibitory activity of furochromone 5 containing thienyl, maybe attributed to the H-bonding which play an important rules in EGFR inhibitory. Also, the oxygen atom of the methoxy group formed a hydrogen bonding with G695 (Gly695) (distance: 2.20 Å, angle: 156.8°). So that, they predict binding free energy including the intermolecular energy and torsional free energy were used as the criterion for ranking. This molecular docking result and biological assay data, suggesting that compound 5 has a potential EGFR inhibitor and the binding free energy is −11.86 kcal/mol, compared to compounds (1–4) in the range of −13.29 to −14.62 kcal/mol.

Three compounds, i.e., 3, 4, and 5, were found that may be promising candidates for further investigation. The main feature shared by these three potential inhibitors as well as the information of the involved side chains of SARS Cov Mpro may provide useful insights for the development of potent inhibitors against SARS enzyme.

Furthermore, the intermolecular hydrogen bonds and electrostatic interaction, whose effects have already been counted in the binding energy, were also investigated in order to find useful information for drug design for compound 5.

Anti-proliferative activity of compounds (2–5) was studied in vitro on a panel of human tumour cell line (MCF-7), which overexpresses EGFR, as shown in Figure 3, out of the top compounds 4 and 5, which have potent inhibitor activity of EGFR presented high anti-proliferative activity against MCF-7 with IC₅₀ of 0.15 and 0.08 µM, and against Hela with IC₅₀ of 0.26 and 0.09 µM indicating that furochromones 1–5 having thienyl ring were potent inhibitor of EGFR as antitumor agents. In particular, compound 5 has demonstrated significant inhibitor activity in tumour growth inhibition and displayed favourable EGFR inhibitor.

Figure 3.  Anti-proliferative activity against MCF-7 and Hela for compounds 1–5. The intensity of the developed colour measured by reading optical absorbance at 450 nm using a microplate reader FLUOstar OPTIMA ^a(IC50 compound concentration required to inhibit tumour cell proliferation by 50%. ^bHuman breast cell line (MCF-7). ^cHuman cervix cell line (HELA).

Molecular modelling study of EGFR inhibitor and binding conformation

Molecular modelling study was initiated in order to support the assumed mode of action for tested compounds and optimize a reliable model for predicating novel effective anti-tumour hits. Docking study was carried out for the target compounds into EGFR using Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA, USA). The crystal structure of the enzyme and erlotinib (1M17) 8 (Scheme 3) was obtained from protein data bank PDB⁶⁰ since erlotinib mimics ATP and binds to the ATP binding region of the kinase active site. Therefore, the synthesized compounds 2–5 were modelled by positioning them in the erlotinib binding site⁶¹.

Scheme 3.  The Model compounds of (6a-e), (7a-g) and erlotinib (1M17) (8) which obtained from protein data bank (PDB)58,59.

The synthesized compounds 2–5 similitude to gefitinib and erlotinib were the first drug of this type. Erlotinib specifically targets of the EGFR tyrosine kinase, which is highly expressed and occasionally mutated in various forms of cancer. It binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the receptor⁶². For the signal to be transmitted, two members of the EGFR family need to come together to form a homo-dimer. These then use the molecule of ATP to auto-phosphorylate each other, which causes a conformational change in their intracellular structure, exposing a further binding site for binding proteins that cause a signal cascade to the nucleus. By inhibiting the ATP, auto-phosphorylation is not possible and the signal is stopped, as shown in Figure 4.

Figure 4.  Lead compound erlotinib (green colour) (A) and khellin 1 (B): the proposed binding mode of compounds (A) and (B) in the ATP binding site of EGFR resulting from docking Energy is −10.86 and −11.38 respectively. The most important amino acids are shown together with their respective numbers.

Interactive docking using CDOCKER protocol was carried out for all the conformers of each compound of 1–5 to the selected active site and each docked compound was assigned according to binding mode. The score according to its binding mode onto the binding site⁶³ that predicted binding energies and the corresponding experimental values as outlined in the Figure 2. Alignment study of docked compound 3 and erlotinib with the binding pocket of EGFR, as shown in Figure 5 revealed that

Figure 5.  2(A), 5(B) and 4(C): the proposed binding mode of Molecular docking modelling of compounds 2(A), 4(C), 5(B) and 3(E,F) with EGFR kinase: for clarity, only interacting residues are displayed in the ATP binding site of EGFR resulting from docking respectively. The most important amino acids are shown together with their respective numbers. Compound 5 form three hydrogen bonds one acceptors with N-H group of Met769 and two donors one with carboxylate oxygen of Asp 331 and one of the carbonyl oxygen of Lys 721. (D): Alignment of docked compound 5 (purple colour) and erlotinib (green colour) in the ATP binding site of EGFR, shows amino acids in contact in the same position. The H-bond is displayed as line. Compound 5 is nicely bound to the EGFR kinase with its N–H group project toward the side chain carbonyl group of D831 (Asp831), forming a more optimal H-bond interaction. Also, the oxygen atom of the methoxy group of compound C5 forms hydrogen bond with G697 (Gly695).

• (i) Benzofuran ring system of compound 5 was perfectly aligned with quinazoline nucleus of ligand erlotinib

• (ii) Methylpyrane moiety of 5 superimposed with 4-anilino substituent of erlotinib

• (iii) Both the erlotinib and compound 5 make the same hydrogen bonding interaction with Met769.

Additionally, compound 5 makes another two hydrogen bonding, one of hydrogen bonding donor interaction with carboxylate oxygen of Asp 331 and the other hydrogen bonding donor with carbonyl oxygen of Lys 721.

In other words, through docking study it could be concluded that the benzofuran ring system is responsible for fitting the tested structures (1–5) in an appropriate position occupied by the quinazoline nucleus of erlotinib (potent EGFR inhibitor). All the target compounds made hydrogen bonding acceptor interaction with N-H group of Met 769 which is vital for the inhibitor activity. However, the heteroaryl group fused to the benzofuran ring system of the tested compounds 1–5 played an important role in optimizing the docking process. These attained observations seem similar to the mentioned ones due to SAR controlling factors. Docking results provided useful information in understanding the binding mode and structural features of the target and chemical features of the ligands. This was extended to the successful designing of highly active analogs of khellin derivatives as EGFR inhibitor.

Biological activity study

The newly synthesized compounds 2–5 were evaluated in vitro as EGFR inhibitor as well as the in vitro study against 59 different human tumour cell lines.

Pharmacological: primary single high dose (10⁵ M) full NCI 60 cell panel in vitro assay

All the selected compounds submitted to National Cancer Institute (NCI) for in vitro anticancer assay were evaluated for their anticancer activity. Primary in vitro one dose anticancer assay was performed in full NCI 57 cell panel representing leukemia, melanoma and cancers of lung, colon, brain, breast, ovary, kidney and prostate in accordance with the protocol of the NCI, USA. The compounds were added at a single concentration (10⁵ M) and the culture was incubated for 48 h. End point determinations were made with a protein binding dye, sulforhodamine B. Results for each compound were reported as a mean graph of the percent growth of the treated cells when compared to the untreated control cells such as compounds 2 (NSC 751491), compounds 4 (NSC 751492) and 5 (NSC 751493).

In vitro anti-tumour activity

Antitumor activity screening of the new synthesized compounds 2–5, adopted by developmental therapeutics program of National Cancer Institute (Bethesida, Maryland, USA), at a dose of 10 mM utilizing 57 different human tumour cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate and kidney was carried out according to the previously reported standard procedure^64–67. The results obtained as shown in Figure 6 represent percentage growth of the tumour cell lines treated with compounds under investigation relative to control cell experiments. From the observed data it has been noticed that, the tested compounds reflect mild activity against most of the used human tumour cells. However, compounds 3 (NSC751493) and 4 (NSC751492) reveal considerable anti-tumour properties against CNS (SNB-75), renal (TK-10), ovarian (IGROV1), colon (HCT-15), breast (T-47D), considering cell line growth inhibition with >50% at a concentration of 10 mM usually seems a noticeable activity.

Figure 6.  The anti-tumour properties of the tested compounds at a dose of 10 mM utilizing human tumour cell lines.

EGFR inhibition activity

Chemotherapy is a major approach for both localized and metastasized cancers^68,69. So that, the newly synthesized compounds were screened for their inhibitor activities against EGFR on human breast carcinoma cell line (MCF-7) and human cervical carcinoma cell line (HELA) in comparison with the activity of the known EGFR inhibitor of erlotinib as a reference drug. The inhibitory activities of the tested compounds were expressed as IC₅₀ mg/mL. The screening results are compiled in Figures 1, 2 and 3 revealed that, the synthesized compound 2–5 having a new potent EGFR inhibitors, e.g. compound 5 compared to erlotinib 8. Khelline a naturally occurring furochromone designing for potential EGFR kinase inhibitors have been discovered and some of them exhibited significant EGFR inhibitor activity.

Conclusions

Khelline a naturally occurring furochromone (1–5) designing for potential EGFR kinase inhibitors have discovered and some of them exhibited significant EGFR inhibitory activity. Compound 5 exhibited the most potent EGFR inhibitory activity with IC₅₀ of 0.04 µM. Antiproliferative assay results indicated that these furochromone derivatives 2–5 own high antiproliferative activity against MCF-7 and Hela.

Experimental

Chemistry

Melting points were determined on a Gallenkamp apparatus and are uncorrected. IR spectra were recorded using Perkin Elmer FT-IR 1650 and Pye-Unicam SP300 infrared spectrophotometers. The ¹H and ¹³C NMR spectra were taken on a Varian VXP-300 (300 MHz and 75 MHz respectively) in DMSO-d6, internal standard TMS, ³¹P NMR spectra (122 MHz), external standard 85% H₃PO₄. It was measured at department of chemistry, University of Liverpool, UK. Elemental analyses were carried out at the Microanalytical Laboratory of Cairo University, Giza, Egypt.

2-Amino-6,10-dimethoxy-4-methyl-5a,6,10,10a-tetrahydro-4H-5,7-dioxa-3-thia-dicyclopenta[a,g] naphthalene-1-carbonitrile (2)

A mixture of khellin 1 (1.32 g, 5 mmol), sulphur (S₈) (0.16 g, 5 mmol) and malononitrile (0.33 g, 5 mmol) in the presence of Et₃N 0.5 mL in absolute ethanol 25 mL of was refluxed for 3 h. After cooling, the reaction mixture was filtered off and the solid isolated by filtration which the crystalline material from ethyl acetate to give, 0.25 g (20% yield) Brown crystals of compound 2, mp 182–184°C.; IR (Vaseline oil, cm⁻¹): 3325, 2214, 1720 (C=O), 1610, 1580, 1550 w (arom.). ¹H NMR δ 2.36 (s, 3H, CH₃), 2.50 (m, 2H, 2CH), 3.92 (s, 3H, CH₃), 4.09 (s, 3H, CH₃), 6.05 (s, 1H, CH), 6.85 (s, 2H, CH), 7.02-7.21 (m, 2H, CH), 8.09 (s, 2H, NH₂). ¹³C NMR (DMSO-d₆) δ 39.30, 39.93, 40.14, 61.17, 61.75, 105.10, 109.95, 113.12, 129.32, 146.65, 146.69, 148.23, 152.07, 164.21, 179.58. Anal. Calcd for C₁₇H₁₈N₂O₄S (346.4): C, 58.94; H, 5.24; N, 8.09 S, 9.26 Found: C, 58.55; H, 5.20; N, 8.12; S, 9.18.

3-Imino-4,8-dimethoxy-10-methyl-2,2-disulfanyl-furo[2,3-g]thieno[2,3-c]chromene,thieno[3,2-d][1,2]azaphosphol-2-ium (3)

A mixture of compound 2 (1.00 g, 3 mmol) and P₂S₅ (2 g, 9 mmol) in 25 mL of pyridine was stirred and refluxed for 3 h. After cooling, the reaction mixture was filtered off with dry isolated solid and re-crystallization by ethyl acetate to give, 0.80 g (60% yield ) black brown crystals of compound 3, mp 160°C decomposed. IR (KBr) ν 3325, 1462 cm⁻¹. ¹H NMR δ = 2.30 (s, 3H, CH₃), 4.10 (s, 6H, 2-0CH₃), 4.25 (s, 1H, CH), 6.04 (s, 1H, CH), 6.96 (s, 1H, CH), 7.26-7.27 (d, 2H, CH), 7.83-7.87 (m, 1H, CH), 8.08 (s, 1H, CH), 8.11-8.12 (d, 1H, CH), 8.34-8.36 (s, 1H, NH), 8.80-8.82 (m, 1H, NH). ¹³C-NMR (75 MHz, DMSO-d₆) δ 39.79, 40.35, 61.24, 105.10, 105.27, 119.86, 120.14, 124.14, 126.10, 129.53, 147.81, 147.14, 147.27, 148.07, 153.73, 164.19, 202.12. ³¹P NMR (122 MHz, DMSO-d₆) 0.96.; Anal. Calcd for C₁₇H₁₉N₂O₄PS₄- (474.58): C, 43.02; H, 4.04; N, 5.90; S, 27.03 Found: C, 43.14; H, 4.33; N, 6.03; S, 27.04.

5,9-dimethoxy-11-methyl-furo[2,3-g]thieno[2,3-c]chromene- thieno[2,3-d]pyrimidine-2,4-dithiol (4)

A mixture of furo[2,3-g]thieno[2,3-c]chromene-3-carbonitrile 2 (1.00 g, 3 mmol) and CS₂ (10 mL) in the presence of dry pyridine 25 mL was stirred and refluxed for 10 h and allowed to stand at RT for 2 days, the mixture was triturated with aqueous ethanol (50 mL), cooling, the reaction mixture was filtered off. The precipitate solid was filtered collected and crystallized from ethyl acetate to give 0.85 g (50% yield) reddish brown solid of compound 3, mp >300°C decomposed. IR (KBr) ν 1377 cm⁻¹. ¹H NMR δ 2.43(s, 3H, CH₃), 3.98 (s, 3H, CH₃), 4.16(s, 3H, CH₃), 6.12 (s, 1H, CH), 7.27-7.28 (d, 1H, CH), 7.80-7.83 (s, 1H, CH), 8.29-8.28 (d, 2H, CH), 8.82-8.83 (m, 1H, CH), 10.02 (s, 1H, CH), 10.70 (d, 1H, CH), 12.87 (s, 2H, SH). ¹³C NMR (DMSO-d₆) δ 39.79, 40.35, 61.24, 105.10, 105.27, 119.86, 120.14, 124.14, 126.10, 129.53, 147.81, 147.14, 147.27, 148.07, 153.73, 164.19, 202.12. Anal. Calcd for C₁₈H₁₈N₂O₄S₃ (422.54): C, 51.16; H, 4.29; N, 6.63; S, 22.77 Found: C, 51.20; H, 4.32; N, 6.70; S, 22.89.

5,9-Dimethoxy-2,11-dimethyl-furo[2,3-g]thieno[2,3-c]chromene-thieno[2,3-d]pyrimidine-4-one (5)

A mixture of acetic anhydride/pyridine mixture (30 mL) (2:1 v/v) was added into furo[2,3-g]thieno[2,3-c]chromene-3-carbonitrile 2 (1.00 g, 3 mmol) and heated for 15 h. the mixture was cooling and poured into an ice water the solid product formed was filtered off and washed several times with cold water and recrystallized from acetic acid to give 0.90 g (58% yield) reddish brown solid of compound 3, mp >300°C decomposed. IR (KBr) ν3296,cm⁻¹. ¹H NMR δ 2.262 (s, 6H, 2CH₃), 4.04 (s, 3H, CH₃), 4.21(s, 3H, CH₃), 6.18 (s, 1H, CH), 7.33 (m, 4H, 4CH), 8.20-8.21 (m, 2H, 2CH), 11.93 (s, 1H, NH), ¹³C NMR (DMSO-d₆) δ 38.88, 39.30, 39.51, 39.71, 39.93, 40.13, 61.19, 87.38, 105.11, 109.96, 112.72, 131.21, 143.49, 146.66, 165.33, 168.94, 171.96, 176.59. Anal. Calcd for C₁₉H₂₀N₂O₅S (388.44): C, 58.75 H, 5.19; N, 7.21; S, 8.25. Found: C, 58.83 H, 5.24; N, 7.27; S, 8.33.

EGFR inhibitory assay

Cellular level of EGFR was quantified by sandwich ELISA in MCF-7 and HeLa cells. The level of EGFR was measured in the homogenate tissue by ELISA. The assay uses the quantitative indirect immunoassay technique that uses polyclonal antibody and biotin-linked polyclonal antibody, both of which are specific against EGFR. Commercially available matched paired antibodies were used (R&D Systems Inc. Minneapolis, MN). Briefly, MCF-7 and HeLa cells were treated for 24 h with 0–50 µg/mL of each tested samples, and then homogenized by three though/freezing cycles. The cell homogenate was coated onto 96-well flat bottom microtiter plate (Griener Labortechnik, Kremsmunster, Austria) in diluents, 50 µL/well and incubated 1 h at 37°C then overnight at 4°C in humidified chamber. Plates were washed three times with washing buffer (PBS/0.05% polyoxyethylene-20; Tween-200) and blocked with 200 µL/well blocking buffer (PBS/0.05%Tween-20/5% FBS) and incubated at 37°C for 1.5 h. The plates were washed three times with washing buffer and incubated with the diluted primary antibody. At the end of the incubation period, the plates were washed three times with washing buffer and diluted second biotin labelled antibody was added for 1 h incubation at 37°C. After washing away any unbound substances, the peroxidase-conjugated streptavidin (Jackson Immunsearch Lab, USA) diluted 1:1000 was added to as 50 µL/well, then the plates were incubated for 1 h at 37°C. After an intensive washing, the enzyme reaction was carried out by adding a 50 µL/well of substrate solution (equal volumes of 3, 3′, 5, 5′-tetramethyl benzidine (TMB; 0.4 g/L) and H₂O₂ (0.02% in citric acid buffer; KPL, Kirkegaard and Perry Lab, Gaithersburg, MD, USA). Colour development was stopped by addition of 50 µL/well of stopping buffer (1 M HCl) (Surechern Products, Needham Marker, and Suffolk, UK). The intensity of the developed colour was measured by reading optical absorbance at 450 nm using a micro plate reader FLUOstar OPTIMA, BMG LABTECH GmbH, Offenburg, Germany).

Antiproliferative activities assay

The antiproliferative activities of compounds 1–5 were determined using a standard (MTT)-based colorimetric assay (Sigma). Briefly, cell lines were seeded at a density of 7 × 10³ cells/well in 96-well microtiter plates (Costar). After 24 h, exponentially growing cells were exposed to the indicated compounds at final concentrations ranging from 0.10 into 100 µg/mL. After 48 h, cell survival was determined by the addition of an MTT solution (10 µL of 5 mg/mL MTT in PBS). After 4 h, 100 µL of 10% SDS in 0.01 N HCl was added, and the plates were incubated at 37°C for a further 18 h; optical absorbance was measured at 570 nm on an LX300 Epson Diagnostic microplate reader. Survival ratios are expressed in percentages with respect to untreated cells. IC₅₀ values were determined from replicates of six wells from at least two independent experiments.

Cell culture

Human breast adenocarcinoma cell line (MCF-7) and human cervical carcinoma cell line (HeLa) were purchased from the American Type Culture Collection (ATCC), VA, USA. Cells were routinely cultured in Dulbeco’s Modified Eagle’s Medium (DMEM). Media were supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, containing 100 units/mL penicillin G sodium, 100 units/mL streptomycin sulphate, and 250 mg/mL amphotericin B. Cells were maintained at sub-confluence at 37°C in humidified air containing 5% CO₂. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37°C. Cells were used when confluence had reached 75%. Before dissolved in medium, compounds were tested for endotoxin using Pyrogent Ultra gel clot assay, and they were found endotoxin free. Compounds were dissolved in DMSO (99.9%) and diluted 1000-fold in the assays. In the cellular experiments, results were compared with DMSO-treated cells of the corresponding concentration. All cell culture material was obtained from Cambrex Bioscience (Copenhagen, Denmark). All chemicals were from Sigma/Aldrich, USA, except mentioned. All experiments were repeated three times.

Molecular docking study

The automated docking studies were carried out using Auto-Dock version 4.0. First, Auto Grid component of the program pre-calculates a three-dimensional grid of interaction energies based on the macromolecular target using the AMBER force field. The cubic grid box of 60 Å size (x, y, z) with a spacing of 0.375 Å and grid maps were created representing 17 the catalytic active target site region where the native ligand was embedded. Then automated docking studies were carried out to evaluate the binding free energy of the inhibitors within the macromolecules. The three-dimensional structures of the aforementioned compounds were constructed using Chemistry 3D ultra 11.0 software [Chemical Structure Drawing Standard; Cambridge Soft corporation, USA (2009)], then they were energetically minimized by using MOPAC with 100 iterations and minimum RMS gradient of 0.10. The Gasteiger-Hückel charges of ligands were assigned. The crystal structures of EGFR (PDB code: 1M17) complex were retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters and ligands were eliminated from the protein and the polar hydrogens and the Kollman-united charges were added to the proteins.

Declaration of interest

The work was financed by a grant (Project 55455/2011) from National Natural Science Foundation of Egypt. The authors report no conflicts of interest in this work.

Acknowledgments

The authors would like to express their gratitude and thanks Dr. Amira M Gamal-Eldeen at National Cancer Institute, Cairo, Egypt for doing the antitumor testing of the new compounds. Our sincere acknowledgments to Chemical Computing Group Inc, 1010 Sherbrooke Street West, Suite 910, Montreal, H3A 2R7, Canada, for its valuable agreement to use the package of MOE 2008.10 software.