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. 2022 Dec 3;7(49):45535–45544. doi: 10.1021/acsomega.2c06219

Thieno[2,3-b]thiophene Derivatives as Potential EGFRWT and EGFRT790M Inhibitors with Antioxidant Activities: Microwave-Assisted Synthesis and Quantitative In Vitro and In Silico Studies

Sanaa A Ahmed ‡, Moumen S Kamel †,*, Moustafa O Aboelez §,*, Xiang Ma ∥, Ahmed A Al-Karmalawy ⊥, Sayed A S Mousa #, Elders Kh Shokr , H Abdel-Ghany †, Amany Belal ∇,○, Mohamed A El Hamd ⧫,††,*, Zafer S Al Shehri ‡‡, Mahmoud Abd El Aleem Ali Ali El-Remaily †,*
PMCID: PMC9753534  PMID: 36530244

Abstract

graphic file with name ao2c06219_0010.jpg

Microwave-assisted synthesis and spectral analysis of certain novel derivatives of 3,4-diaminothieno[2,3-b]thiophene-2,5-dicarbonitrile 1–7 were carried out. Compounds 1–7 were examined for cytotoxicity against MCF-7 and A549 cell lines using the quantitative MTT method, and gefitinib and erlotinib were used as reference standards. Compounds 1–7 were shown to be more active than erlotinib against the two cell lines tested. Compound 2 outperformed regular erlotinib by 4.42- and 4.12-fold in MCF-7 and A549 cells, respectively. The most cytotoxic compounds were subsequently studied for their suppression of kinase activity using the homogeneous time-resolved fluorescence assay versus epidermal growth factor receptor (EGFRWT) and EGFR790M. With IC50 values of 0.28 ± 0.03 and 5.02 ± 0.19, compound 2 was demonstrated to be the most effective against both forms of EGFR. Furthermore, compound 2 also had the best antioxidant property, decreasing the radical scavenging activity by 78%. Molecular docking research, on the other hand, was carried out for the analyzed candidates (1–7) to study their mechanism of action as EGFR inhibitors. In silico absorption, distribution, metabolism, excretion, and toxicity tests were also performed to explain the physicochemical features of the examined derivatives.

1. Introduction

Cancer is a well-recognized disease with a multifactorial nature that is known for uncontrolled cell growth and abnormal cell spread as well.1−3 It is ranked to be the second cause of death all over the world.4,5 Nowadays, there are various techniques to fight cancer such as chemotherapy, radiotherapy, and/or surgery.6−10 The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that is involved in the development and homeostasis of epithelial tissues.11−14 Moreover, the family of EGFR has a vital role in cell signaling through overexpression which is linked to the progress of various human solid malignancies, especially breast cancers and non-small-cell lung cancers.15−18 The use of EGFR inhibitors to block EGFR-mediated signaling pathways is a common approach for the treatment of several malignancies.17−19 There are three generations of EGFR-tyrosine kinase inhibitors available: the first one includes reversible EGFR inhibitors (erlotinib and gefitinib); the second generation includes irreversible EGFR blockers (afatinib and dacomitinib); and the third one includes a wild-type-sparing, irreversible EGFR inhibitor (osimertinib). The fourth generation inhibitors (which include EAI04518 and other non-covalent inhibitors) are now in preclinical testing.20,21 In addition, the EGFR is well established as a biomarker in the case of resistant cancers, as its secondary mutations have been observed to occur under treatment components.22

Since the 18th century, researchers have been interested in the interaction of the well-known electromagnetic fields (EMFs), such as microwave frequency range which forms a part of many identities of the EMFs, and various life processes.23−26 Consequently, the solvent-free approach of microwave-assisted synthesis in conjunction with the availability of a supported catalyst or reagents that could be used in synthesis overcame some principles of green chemistry.27 Recently, several papers have utilized this technique in the synthesis of S-containing heterocycles,28−31 which have important medicinal and materials science applications.

However, based on earlier research on the formation of several new pyrrolo[2,3-b]pyrrole derivatives with significant hypotriglyceridemic effects and antioxidant activity,31−36 using MCF-7, HCT-116, and A549 cancer cell lines the antiproliferative activity and in vitro enzymatic inhibitory activity against both types of EGFR were evaluated.

To summarize, in this work, we synthesized fused thieno[2,3-d]pyrimidines 1–7 and examined if they have good anticancer activity versus EGFRWT and EGFR790M.

2. Results and Discussion

2.1. Synthesis of Pyrimidines, Pyridines, and Azoles

Treatment of 3,4-diaminothieno[2,3-b]thiophene-2,5-dicarbonitrile (TT amino cyano) 1(19,37−39) with formamide, formic acid, carbon disulfide, phenyl isocyanate, or phenyl isothiocyanate gave pyrimidine derivatives 2–6. The suggested reaction mechanism of compounds 2–6 is shown in Figure 1. Their IR spectra showed the loss of Inline graphic group absorption bands in all compounds and new bands appeared in NH, Inline graphic, and Inline graphic groups at 3410–3325, 1645, and 1350 cm–1, respectively. Its 1H NMR spectrum revealed a novel NH signal at Inline graphic 12.85, 7.39, 9.74, and 9.83 ppm for compounds 3, 4, 5, and 6, respectively, and showed aromatic signals at 8.50, 8.33, 7.87, and 7.78 ppm for compounds 2, 3, 5, and 6, respectively. The 13C NMR spectra of compounds 5 and 6 showed new signals for Inline graphic and Inline graphic at δ 165.2 and 175.1 ppm, respectively (Scheme 1).

Figure 1.

Figure 1

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Reaction mechanisms for (a) compound 2, (b) compound 4, and (c) compounds 5 and 6.

Scheme 1. Synthesis of Target Compounds (2–7).

Scheme 1

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2,5-Di(1H-tetrazol-5-yl)thieno[2,3-b]thiophene-3,4-diamine 7 was synthesized via the reaction of compound 1 with sodium azide. Compound 7 was formed via the reaction of [2 + 3] cycloaddition in the presence of copper sulfate pentahydrate.40 A possible mechanism is shown in Figure 2. The nitrile compounds’ nitrogen atoms first form a complex when they are coordinated with CuII, accelerating the cyclization process. The Inline graphic bond of the nitrile molecule and the azide ion easily undergo a [3 + 2] cycloaddition to generate the intermediate. III and IV are enabled by acidic media. The more stable tautomer IV is created as a result of equilibrium. This compound’s IR spectrum revealed its elimination of the CN group. A new signal was discovered in its 1H NMR spectrum due to NH at Inline graphic 6.12 ppm.

Figure 2.

Figure 2

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Mechanism for the synthesis of compound 7.

2.2. Biological Assessments

2.2.1. Cytotoxic Actions In Vitro

The MTT test was used to assess the antiproliferative effect of target compounds 1–7 versus a selection of two cell lines, MCF-7 and A549, in vitro.19−21 Erlotinib and doxorubicin were used as controls in this investigation; in the MCF-7 and A549 cell lines, EGFRWT is abundantly expressed.21,22Table 1 displays the data as IC50 (M). Compounds 1–7 were shown to be more active against MCF-7 and A549 than erlotinib. Compound 2 was found more effective than the standard in MCF-7 and A549 cells by 4.42 and 4.12 times, respectively. Compound 2 was more active than doxorubicin versus MCF-7 and A549 cells. Besides, compound 3 was found efficient versus the MCF-7 cell line.

Table 1. Compounds 1–7 Have Antiproliferative Effects In Vitro vs MCF-7 and A549 Cell Linesa.
compounds IC50 (μM) (n = 3)
1 8.57 ± 0.31 5.09 ± 0.15
2 4.92 ± 0.19 4.69 ± 0.09
3 8.96 ± 0.35 4.19 ± 0.19
4 9.26 ± 0.25 5.67 ± 0.13
5 17.26 ± 0.71 7.54 ± 0.32
6 15.26 ± 0.69 9.27 ± 0.25
7 17.26 ± 0.71 8.27 ± 0.16
erlotinib 21.76 ± 1.85 19.33 ± 1.85
doxorubicin 7.89 ± 0.55 NT
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a

NT: compounds were not examined.

2.2.2. EGFRWT Kinase Inhibitory Assay

Compounds 1–7 have an inhibiting impact on the EGFRWT kinase enzyme and were examined by the homogeneous time-resolved fluorescence (HTRF) measure21,23,24 utilizing erlotinib as the control drug (Table 2 and Figure 3). Compounds 1, 2, and 3 were more effective than erlotinib (0.32 ± 0.05 μM) versus EGFRWT with IC50 values of 0.29, 0.28, and 0.29 μM, respectively. With IC50 values of 0.75 μM, compound 4 was shown to have the same activity as erlotinib. Finally, with IC50 values of 2.02, 2.77, and 3.27 μM, respectively, compounds 5–7 showed moderate activity.

Table 2. In Vitro Enzymatic Inhibitory Effects of Compounds 1–7 vs EGFRWT and EGFR790Ma.
compounds EGFRWT IC50 (μM) (n = 3) EGFR790M IC50(μM, n = 3)
1 0.29 ± 0.05 7.89 ± 0.31
2 0.28 ± 0.03 5.02 ± 0.19
3 0.29 ± 0.09 7.26 ± 0.32
4 0.75 ± 0.03 9.16 ± 0.28
5 2.02 ± 0.12 15.06 ± 0.51
6 2.77 ± 0.05 17.26 ± 0.69
7 3.27 ± 0.16 16.26 ± 0.61
erlotinib 0.32 ± 0.05 NT
gefitinib NT 21.44 ± 0.75
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a

NT: compounds were not investigated.

Figure 3.

Figure 3

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Inhibiting impact on the EGFRWT and EGFR790M for compounds 1–7.

2.2.3. EGFRT790M Kinase Inhibitory Assay

The ability of target compounds 1–7 to inhibit mutant EGFRT790M was explored further when they had substantial IC50 values against EGFRWT. Gefitinib was used as a standard reference. Most of the target compounds can inhibit with IC50 values ranging from 5.02 to 17.26 μM to suppress the EGFRT790M activity, indicating that they are more effective than gefitinib (Table 2). In particular, compound 2 (IC50 = 5.02 ± 0.19 μM), the most potent counterpart, was found to be 4.27 times as powerful than gefitinib (IC50 = 21.44 ± 0.75 μM). The most active analogues were compounds 1, 3, and 4, which have 2.71, 2.95, and 2.34 times the activity of gefitinib, respectively. Finally, the inhibitory effects of compounds 5–7 nearly reached IC50 values of gefitinib (Table 2).

2.2.4. Correlation of Cytotoxicity with EGFRWT Inhibition

EGFRWT has been illustrated to be represented by compounds 1–7. After that, we explored whether blocking EGFRWT had an antiproliferative effect within the two cell lines we examined. Utilizing the GraphPadPrism 8 program, a simple linear regression design was used to plot the target derivative’s activity as EGFRWT inhibitors versus their cytotoxicity. The measured coefficients of determination (R2) exhibit the relationship between EGFRWT inhibition and actuated antiproliferative activity; MCF-7 and A549 showed R2 amounts of 0.833 (P = 0.004) and 0.911 (P = 0.001), respectively (Figure 4A,B).

Figure 4.

Figure 4

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(a,b).Correlation between EGFRWT inhibition and cytotoxicity in MCF-7 and A549 cell lines.

2.2.5. Antioxidant Activity by ABTS Method

Compounds 1–7 were evaluated for antioxidant action utilizing the 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid)diammonium salt (ABTS) strategy.25−28 The ABTS index considers antioxidants’ capacity to scavenge the long-lived ABTS radical cation. To transform ABTS to its radical cation, sodium persulfate is needed. This radical cation absorbs light at 734 nm and gives a blue color. The ABTS radical cation responds with the majority of antioxidants, namely, phenolics, thiols, and vitamin C. During the whole reaction, the blue ABTS radical cation is changed to its colorless neutral form. The reaction can be detected via spectrophotometric analysis.

We can call the previous test the trolox equivalent antioxidant capacity assay. Figure 5 exhibits the percentages of radical scavenging activity inhibited for 1–7 that were studied. The maximum antioxidant activity was found in compounds 2, 3, and 5, repressing radical scavenging activity to 78, 64, and 70%, respectively, while compounds 1, 4, 6, and 7 have average antioxidant activity (39–49%).

Figure 5.

Figure 5

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Radical scavenging activity of 1–7 using the ABTS radical method.

2.3. In Silico Studies

2.3.1. Docking Study

Initially, the binding mode of the co-crystallized HYZ inhibitor was analyzed within the target receptor of EGFR. The results demonstrated their stabilization inside its binding pocket through the formation of two hydrogen bonds with the Met793 amino acid and one Inline graphic–H interaction with Leu844 amino acid. This refers to the great importance of binding both Met793 and Leu844 amino acids to induce an EGFR antagonistic activity. Herein, the most active compounds (1, 2, and 3) were selected for further investigation to describe the binding mode and interactions (Table 3). Compound 1 (S = −5.58 kcal/mol, rmsd = 1.18) formed three hydrogen bonds with Asp855, Lys745, and Met793 at 2.97, 3.32, and 3.41 Å, respectively. Also, it formed one Inline graphic–H bond with Val726 at 4.19 Å. Besides, compound 2 (S = −5.86 kcal/mol, rmsd = 1.79) was additionally fixed with the EGFR binding site after the creation of two hydrogen bonds with Met793 at 2.79 and 3.29 Å and one hydrogen bond with Lys745 at 3.56 Å. Also, it formed two Inline graphic–H bonds with Val726 at 3.63 and 4.37 Å and one Inline graphic–H interaction with Leu718 at 4.57 Å as well.

Table 3. Binding Scores, rmsd, 3D Binding Interactions, and 3D Positioning of the Newly Synthesized Derivatives (1, 2, and 3) inside the Binding Pocket of the EGFR, besides the HYZ Inhibitor (4).

2.3.1.

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a

S: score of a compound within the protein-binding pocket (kcal/mol).

However, compound 3 (S = −6.18 kcal/mol, rmsd = 1.15) indicated three hydrogen bonds formed with Lys745, Met793, and Asp855 at 3.05, 3.10, and 3.28 Å and 3 Inline graphic–H bonds with Leu718, Val726, and Met793 at 3.93, 3.98, and 4.81 Å, respectively. Whereas the docked HYZ inhibitor (S = −11.33 kcal/mol, rmsd = 1.85) bound Met793 amino acid with two H-bonds at 3.13 and 3.19 Å and Lys745 with a Inline graphic–H interaction at 4.13 Å. Based on the above, we can observe that all studied derivatives bound the crucial amino acids within the binding pocket of EGFR (Met793); therefore, they are highly recommended to act as promising EGFR inhibitors. Moreover, they got stabilized through the formation of extra bonds with good binding scores.

2.3.2. Physicochemical and ADME Profile

Our previous studies focused on investigating the physicochemical properties of organic molecules41 due to their importance in getting better insights into molecular polarity, lipophilicity, bioavailability, and so forth. Absorption, distribution, metabolism, and excretion (ADME) features of the produced compounds were studied using the SwissADME Web server; the boiled egg chart for the cluster showed that all of them have low GI absorption and none of them can penetrate the blood–brain barrier (BBB); this may indicate that they will not cause any central nervous system (CNS) side effects due to their low BBB permeability. Additionally, none of them is predicted to be a P-glycoprotein (P-gp) substrate except compounds 2 and 7, so there is a possibility that these two compounds may suffer from drug resistance. The topological polar surface area (TPSA) ranged from 147 to 209 A2 and the logP value ranged from −0.79 to 4.39, as shown in Table 4. All are predicted to have a good bioavailability score at a value of 0.55; also, all agree with the Lipinski rule of five except compound 7 because it has more than five hydrogen bond donors ; additionally, compound 6 showed only one violation due to the value of MlogP that is more than 4.15.

Table 4. Predicted Physicochemical Properties of the Designed Compounds 1–7.
Comp TPSA (A2) logP GI absorbance BBB permeability P-gp substitution H-bond acc. H bond bioavailability score Lipinski
1 156.10 1.66 low no No 2 2 0.55 yes
2 160.08 1.79 low no Yes 4 2 0.55 yes
3 147.98 1.81 low no No 4 2 0.55 yes
4 248.00 4.37 low no No 0 4 0.55 yes
5 179.76 3.95 low no No 4 4 0.55 yes
6 209.80 5.49 low no No 2 4 0.55 yes; 1 violation: MlogP > 4.15
7 205.09 –0.79 low no yes 6 9 0.55 yes; 1 violation: NH or OH > 5
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3. Experimental Section

3.1. General Chemistry

Merck, Aldrich, and Fluka supplied all of the chemicals utilized in this experiment. NMR spectral data were generated using the attenuated total reflection (ATR) technique on a Bruker spectrophotometer, and infrared spectra were recorded on a Bruker Alpha-Platinum ATR-FT-IR spectrometer (400 MHz and 100 MHz for 1H and 13C NMR, respectively). Chemical changes were quantified in parts per million (ppm) in proportion to an internal reference tetramethylsilane.

The coupling constants (J) for singlet, doublet, triplet, quartet, and multiplet for 1H NMR were given in hertz and denoted as (s) for singlet, (d) for doublet, (t) for triplet, (q) for quartet, and (m) for multiplet. The solvent employed was DMSO-d6. The results of the C, H, N, and S microanalyses were all within 4 percent of the theoretical results.

3.1.1. General Procedure for Preparation of Compounds 2 and 3

Compound 1 (0.219 g, 1 mmol) was microwaved for 10 min at 900 W with excess formamide (10 mL) or formic acid (10 mL). The mixture was put into ice water after cooling for a while. The resulting solid product was filtered, washed with water, left to dry, and crystallized using ethanol.

3.1.1.1. Pyrimido[4″,3″:4′,5′]thieno[3′,2′:4,5]thieno[3,2-d]pyrimidine-4,7-diamine (2)

Yield 75%, yellowish powder, mp > 350 °C; FTIR (KBr) ν cm–1: 3345–3276 (NH2) and 3093 (aromatic); 1H NMR δ ppm: 8.50 (s, 2H, CH pyrimidine) and 7.49 (s, 4H, NH2); 13C NMR δ ppm: 160.2, 158.4, 152.8, 140.1, 124.5, and 107.4; Anal. Calcd for: C10H6N6S2 (274): C, 43.78; H, 2.20; N, 30.64; S, 23.37. Found: C, 43.67; H, 2.13; N, 30.60; S, 23.29.

3.1.1.2. Pyrimido[4″,3″:4′,5′]thieno[3′,2′:4,5]thieno[3,2-d]pyrimidine-4,7(3H,8H)-dione (3)

Yield 70%, yellow powder, mp > 350 °C; FTIR (KBr) ν cm–1: 3599–3514 (NH), 3053 (aromatic) and 1661 (CO); 1H NMR δ ppm: 12.85 (s, 2H, OH) and 8.33 (s, 2H, CH pyrimidine); 13C NMR δ ppm: 161.7, 147.3, 145.8, 144.2, 134.2, and 120.1; Anal. Calcd for C10H4N4O2S2 (276): C, 43.47; H, 1.46; N, 20.28; S, 23.21. Found: C, 43.40; H, 1.38; N, 20.16; S, 23.15.

3.1.2. General Procedure for Preparation of Compounds 4–6

In pyridine (15 mL), a combination of compound 1 (0.219 g, 1 mmol) and (2 mmol) carbon disulfide (0.152 g), phenyl isocyanate (0.239 g), phenyl isothiocyanate (0.270 g), or ethyl cyanoacetate (0.260 g) was microwaved at 1000 W for 15 min. After chilling in ice water, the precipitate was filtered, rinsed, dried, and crystallized using ethanol/DMF.

3.1.2.1. Pyrimido[4″,3″:4′,5′]thieno[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,4,7,9(1H,3H,8H,10H)-tetrathione (4)

Yield 75%, orange powder, mp > 330 °C; FTIR (KBr) ν cm–1: 3410–3385 (NH); 1H NMR δ ppm: 8.63 (s, 2H, NH) and 7.39 (s, 2H, NH); 13C NMR δ ppm: 164.7, 161.8, 149.9, 136.3, 129.2, and 124.3; Anal. Calcd for C10H4N4S6 (372): C, 32.24; H, 1.08; N, 15.04; S, 51.64. Found: C, 32.08; H, 1.19; N, 15.09; S, 51.56.

3.1.2.2. 1,8-Diimino-2,7-diphenyl-1,4,7,8-tetrahydro-2H,5H-9,10-dithia-2,4,5,7-tetraaza-indeno[1,2-a]indene-3,6-dione (5)

Yield 85%, yellow powder, mp > 350 °C; FTIR (KBr) ν cm–1: 3320–3278 (NH), 3050 (aromatic) and 1645 (2CO); 1H NMR δ ppm: 9.74 (s, 2H, NH), 7.87–7.14 (m, 10H, Ar-H), and 6.78 (s, 2H, NH); 13C NMR δ ppm: 165.3, 165.2, 153.0, 140.1, 129.2, 152.2, 122.1, 118.7, 116.1, and 106.2; Anal. Calcd for C22H14N6O2S2 (458): C, 57.63; H, 3.08; N, 18.33; S, 13.98. Found: C, 57.46; H, 3.15; N, 18.22; S, 13.89.

3.1.2.3. 4,7-Diimino-3,8-diphenyl-3,4,7,10-tetrahydropyrimido[4″,3″:4′,5′]thieno[3′,2′:4,5]thieno[3,2-d]pyrimidine-2,9(1H,8H)-dione (6)

Yield 83%, yellowish powder, mp > 350 °C; FTIR (KBr) ν cm–1: 3307, 3207, 3156 (4NH), and 3047 (aromatic); 1H NMR δ ppm: 9.83 (s, 2H, NH), 7.78–7.01 (m, 10H, Ar-H), and 6.95 (s, 2H, NH); 13CNMR: δ 175.1, 154.8, 150.0, 138.9, 136.6, 129.8, 128.8, 124.1, 122.8, and 122.6 Anal. Calcd for C22H14N6S4 (490): C, 53.86; H, 2.88; N, 17.13; S, 26.14. Found: C, 53.97; H, 2.76; N, 17.07; S, 26.22.

3.1.3. General Procedure for Preparing Compound 7

Compound 1 (0.219 g, 1 mmol), sodium azide (0.130 g, 2 mmol), and copper sulfate pentahydrate (0.320 g, 2 mmol) were kept in DMSO (5 mL) for 10 min before heating for 3 h at 140 °C. After TLC, the reaction mixture was left to cool before being processed with 10 mL of HCl and then with 10 mL of ethyl acetate. The resultant organic layer was separated, washed with water (20 mL), dried, concentrated, and crystallized from n-hexane/ethyl acetate (1:1).

3.1.3.1. 2,5-Di(1H-tetrazol-5-yl)thieno[2,3-b]thiophene-3,4-diamine (7)

Yield 70%, green powder, mp > 350 °C; FTIR (KBr) ν cm–1: 3326–3217 (NH2, NH); 1H NMR (DMSO-d6): δ 6.12 (s, 2H, NH) and 4.27 (s, 4H, NH2); 13C NMR δ ppm: 161.0, 152.9, 137.5, 129.3, 123.8, and 118.6; Anal. Calcd for C8H6N10S2 (306): C, 35.40; H, 2.31; N, 41.29; S, 21.00. Found: C, 35.49; H, 2.17; N, 41.07; S, 21.09.

3.2. Biological Evaluation

3.2.1. In Vitro Antiproliferative Activity

Using the MTT test method, the antiproliferative properties of the chosen compounds 1–7 were evaluated in vitro against MCF-7 and A549 cell lines. VACSERA (Cairo, Egypt), a biological supply and vaccine holding agency, obtained ATCC (American-Type Culture Collection) cell lines. The antiproliferative property was determined using the following procedures. Human cancer cell lines were implanted in 96-well plates at a dose of 3–8 × 103 cells per well. The wells were then incubated at 37 °C for 12 h in a 5% CO2 incubator. To determine the DMSO level, each well’s culture medium was replaced with 0.1 mL of new medium containing graded quantities of the target compounds. Following 2 days of hatching time, the cells were matured in 100 μL of MTT solution (5 μg mL–1) for 4 h in each well. After dissolving MTT-formazan crystals in 100 μL of DMSO, the absorption intensity was determined photometrically at 490 nm using an automated ELISA reader system (TECAN, CHE). The IC50 values were then determined using nonlinear regression fitting models (GraphPad Prism, version 5) (n = 3, duplicate trials, reported as mean SD).29,30

3.2.2. EGFRWT and EGFRT790M Kinase Inhibitory Assay

When significant IC50 values versus target cell lines were identified, the inhibitory activities of derivatives 1–7 versus both EGFRWT and EGFRT790M were studied more. In this study, the HTRF test with EGFRWT and EGFRT790M (Sigma) was performed. For the first 5 min, the compounds (1–7) were incubated in an enzymatic buffer with EGFRWT and/or EGFRT790M and their substrates. To start the enzymatic activity, 1.65 M ATP was allowed to react. The reaction ran for half an hour at 210 K. When EDTA-containing test reagents were introduced, the procedure was halted. After a 1 h detection period, the IC50 values were computed by GraphPad Prism 5.0 program. Each concentration was evaluated using three different ways.37,38

3.2.3. Anti-oxidant Activity (ABTS Method)

Derivatives from 1 to 7’s antioxidant activity were calculated using the ABTS technique. The ABTS radical cation stock solution was formed by adding ammonium peroxodisulfate (NH4)2S2O8 solution (0.2 mL, 65 mmol/dm3) with ABTS solution (50 mL, 1 mmol dm3, prepared in 0.1 mol dm3 buffer solution of phosphate pH 7.4). After overnight sitting, the absorbance at 734 nm (AABTS) was measured in a sample cell by adding the ABTS radical cation stock solution (0.5 mL) with a buffer solution of phosphate (2 mL, pH 7.4). After 1 min, the test compounds (0.5 mL, 0.5 mmol) were added to the sample cell, quickly mixed, and the absorbance at 734 nm (compound) was determined.

The absorbance reduction resulting from the extinction of trolox as the reference was evaluated. Same conditions were used for each dosage of trolox (50–600 mol/L), and the calibration graph belonging to the trolox absorption reduction (A = AABTS – ATrolox) versus the concentration of trolox were plotted to obtain a linear regression equation. The antimicrobial effect of the chosen compounds was measured by trolox equivalents using this calibration curve.

3.3. In Silico Studies

3.3.1. Docking Investigation

A molecular docking investigation was carried out using MOE 2019.01241,42 to propose and describe the proposed technique of work for the newly investigated derivatives (1–7) as promising EGFR inhibitors. The co-crystallized HYZ inhibitor (8) was also added as a reference standard to the prepared database.

3.3.1.1. Validation of the Docking Process

The co-crystallized HYZ inhibitor was docked inside its binding site within the EGFR target protein to validate the docking results of the tested compounds. This was done by observing both the rmsd (root-mean-square deviation) value and the obtained binding mode of the docked HYZ inhibitor as well.43−46 In brief, a notable similar binding mode (superimposition) between the docked and native poses of the HYZ inhibitor (represented in red and green colors, respectively) with a low RMSD of 1.61 indicated a satisfactory valid performance for the applied docking program (Figure 6).

Figure 6.

Figure 6

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2D and 3D pictures for the superimposition between the co-crystallized HYZ inhibitor and the docked one (represented in green and red colors, respectively).

3.3.1.2. Structural Preparation of the Target Compounds (1–7)

The new target compounds (1–7) were sketched individually, drawn using the ChemDraw program, transferred to the working window of the MOE, and docked by the previously revised processes.47−50 Both the prepared derivatives (1–7) and the HYZ inhibitor (8) were introduced into a single database for the docking step.

3.3.1.3. Structural Preparation of the EGFR Protein Receptor

The specific EGFR receptor was obtained from the Protein Data Bank Web site (ID: 2RGP(51)). It went through the same preparatory process that was previously mentioned in detail.52,53

3.3.1.4. Docking into the EGFR Binding Site

A generic docking operation was initiated at the ligand’s location, utilizing the prepared database and all of the previously specified software parameters.54−56 Finally, the best pose was selected for each compound to be further investigated according to the obtained scores, binding mode similarity, and RMSD values.57−59

3.3.2. Physicochemical and ADME Profile

Compounds were drawn by ChemDraw; their chemical names were generated, and then transformed to SMILES using the OPSIN Web server. After getting the compounds’ SMILES, they were submitted to the SWISSADME network server to obtain their physicochemical profile.

4. Conclusions

TT amino cyano derivatives (1–7) were synthesized. All derivatives were studied for antiproliferative activities in vitro, which showed a high inhibitory impact on MCF-7 and A549 cell lines. Compound 2 was shown to be 5.54, 4.41, and 4.12 times as active as erlotinib in MCF-7 and A549 cells, respectively. Investigations were performed to explain the viewpoint and understand the inhibitory characteristics of the derivatives versus EGFRWT and EGFRT790M. Accordingly, inhibitory activities versus the two selected isoforms, EGFRWT and EGFRT790M, were investigated for the obtained compounds (1–7). Compound 2 exhibited the highest levels of EGFR inhibitory activity, with IC50 values of 0.28 ± 0.03 and 5.02 ± 0.19 μM, respectively, versus EGFRWT and EGFRT790M. It had the strongest antioxidant activity indicated by 78% inhibition of radical scavenging activity. Moreover, molecular docking experiments revealed that all of the candidates tested bound the critical amino acid within the binding pocket of EGFR (Met793); therefore, they are highly recommended as promising EGFR inhibitors. They got stabilized through the formation of extra bonds with good binding scores. Finally, the obtained results suggested that the obtained compound (2) could be selected as a promising EGFR inhibitor and recommended that further advanced preclinical studies are needed to prove this effect.

Acknowledgments

The authors would like to thank the Deanship of Scientific Research at Shaqra University for supporting this work. Also, the authors are deeply grateful to Sohag University in Egypt for supporting and facilitating this study. The authors would like to acknowledge School of Pharmacy, Huazhong University of Science and Technology, Wuhan, China, for supporting this work.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c06219.

  • 1H NMR and 13C NMR spectral data for compounds 2–7 (PDF)

Author Contributions

S.A.A.A., M.S.K., M.O.A., and X.M.: Conceptualization, methodology, investigation, resources, and writing the original draft. X.M, A.A.A.-K., S.A.S.M., E.K.S., and M.A.S.A.: Writing and editing. H.A.-G.: Data curation, writing, review, and editing. A.B.: Conceptualization, methodology, and visualization. M.A.E.H. and Z.S.A.S.: Formal analysis, software, data curation, method validation, investigation, writing, and editing. M.A.E.A.A.E.R.: Investigation, writing, and editing. All authors have read and agreed to the published version of this manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao2c06219_si_001.pdf (1.7MB, pdf)

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