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Article

New Anticancer Theobromine Derivative Targeting EGFRWT and EGFRT790M: Design, Semi-Synthesis, In Silico, and In Vitro Anticancer Studies

by
Eslam B. Elkaeed
1,
Reda G. Yousef
2,
Hazem Elkady
2,
Aisha A. Alsfouk
3,
Dalal Z. Husein
4,
Ibrahim M. Ibrahim
5,
Ahmed M. Metwaly
6,7,* and
Ibrahim H. Eissa
2,*
1
Department of Pharmaceutical Sciences, College of Pharmacy, AlMaarefa University, Riyadh 13713, Saudi Arabia
2
Pharmaceutical Medicinal Chemistry & Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt
3
Department of Pharmaceutical Sciences, College of Pharmacy, Princess Nourah bint Abdulrahman University, Riyadh 11671, Saudi Arabia
4
Chemistry Department, Faculty of Science, New Valley University, El-Kharja 72511, Egypt
5
Biophysics Department, Faculty of Science, Cairo University, Cairo 12613, Egypt
6
Pharmacognosy and Medicinal Plants Department, Faculty of Pharmacy (Boys), Al-Azhar University, Cairo 11884, Egypt
7
Biopharmaceutical Products Research Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications (SRTA-City), Alexandria 21934, Egypt
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(18), 5859; https://doi.org/10.3390/molecules27185859
Submission received: 31 July 2022 / Revised: 28 August 2022 / Accepted: 6 September 2022 / Published: 9 September 2022
(This article belongs to the Special Issue Molecular Modeling: Advancements and Applications II)
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Versions Notes

Abstract

:
Based on the pharmacophoric features of EGFR inhibitors, a new semisynthetic theobromine-derived compound was designed to interact with the catalytic pocket of EGFR. Molecular docking against wild (EGFRWT; PDB: 4HJO) and mutant (EGFRT790M; PDB: 3W2O) types of EGFR-TK indicated that the designed theobromine derivative had the potential to bind to that pocket as an antiangiogenic inhibitor. The MD and MM-GBSA experiments identified the exact binding with optimum energy and dynamics. Additionally, the DFT calculations studied electrostatic potential, stability, and total electron density of the designed theobromine derivative. Both in silico ADMET and toxicity analyses demonstrated its general likeness and safety. We synthesized the designed theobromine derivative (compound XI) which showed an IC50 value of 17.23 nM for EGFR inhibition besides IC50 values of 21.99 and 22.02 µM for its cytotoxicity against A549 and HCT-116 cell lines, respectively. Interestingly, compound XI expressed a weak cytotoxic potential against the healthy W138 cell line (IC50 = 49.44 µM, 1.6 times safer than erlotinib), exhibiting the high selectivity index of 2.2. Compound XI arrested the growth of A549 at the G2/M stage and increased the incidence of apoptosis.

1. Introduction

World Health Organization, WHO, statistics indicate that cancer will become the major cause of death within a few years [1]. In response, developing safe and effective cancer therapies that bind with a specific molecular target and destroy cancer cells represents a challenging problem for scientists [2]. Angiogenesis contributes to tumor growth and reproduction. Hence, stopping angiogenesis is considered one of the possible strategies to treat cancer [3]. Angiogenesis and cancer cell growth depend greatly on the epidermal growth factor receptor (EGFR) [4]. Overexpression of EGFR promotes cellular proliferation, differentiation, and survival through downstream signaling pathways. The EGFR gene promotes the growth of various solid tumors and is elevated in multiple types of cancer [5]. Increased EGFR expression was proven to be associated with a reduced survival rate, and acted as a strong prognostic indicator [6]. EGFR receptors are overexpressed in cancer more than in normal cells, allowing researchers to target them as a cancer-fighting strategy [7].
Natural products have historically been the cornerstone of drug development [8,9]. Recently, natural products accounted for almost one-third of newly approved FDA drugs in the period 1981–2014 [10]. Xanthines, as well as xanthine derivatives, are an extremely interesting category of compounds in the field of anticancer drug discovery as they expressed various antimutagenic activities against different cancer types such as ovarian [11], prostate [12], leukemia [13], and breast [13] cancers. First discovered in 1841 [14], theobromine is a xanthine alkaloid (3,7-dimethylxanthine) found primarily in Theobroma cacao, “chocolate”, as well as in a variety of foods such as tea leaves [15]. It was first synthesized in 1882 [16]. Interestingly, theobromine expressed promising activities against colon cancer through in vitro [17] as well as in vivo [18] examinations. Additionally, theobromine showed anti-carcinogenic activity by inhibiting the DNA synthesis in cancer cells [19]. Further, theobromine inhibited the growth of glioblastoma multiforme in vitro [20]. Remarkably, theobromine could prevent angiogenesis in lung cancer in an in vivo study [21] as well as it exhibits promising anti-angiogenic activity via the inhibition of the vascular endothelial growth factor (VEGF), in vivo and in vitro, in ovarian cancer [22].
The semi-synthesis of natural products can aid in discovering more potent drugs and repurposing, as well as improving drug-likeness, pharmacokinetics, and pharmacodynamics [23].
Computational (computer-based, computer-aided) chemistry is used to explore the interactions of potential drugs with biomolecules using theoretical ideas and computer techniques [24]. Our team applied in molecular docking [25,26], molecular design [27], toxicity [28,29], ADMET [30,31], DFT [32,33], structural similarity [34], MD [35], and pharmacophore [36] evaluation.
The clinically approved EGFR tyrosine kinase-inhibitors (EGFR TKIs) (Figure 1) have some problems. The anticancer effect of the 1st-generation EGFR-TKI as erlotinib I [37] was decreased due to the drug resistance acquired by a certain mutation (EGFRT790M) [38]. In addition, erlotinib was reported to exert life-threatening lung disease [39]. Although overcoming the drug resistance induced by EGFRT790M, the 2nd-generation EGFR-TKIs (as neratinib II [40]) showed poor clinical patient outcomes due to a low maximal-tolerated-dose [41,42]. Olmutinib III, the 3rd-generation EGFR-TKIs, showed improved activities towards EGFRT790M. Unfortunately, two cases of toxic epidermal necrolysis (one of them was a fatal case) and a case of Stevens–Johnson Syndrome were recorded by Olmutinib [43]. Compound IV was generated by Traxler et al. [44] and showed excellent efficacy in inhibiting EGFR TK. Compound V is also an example of 1H-pyrazolo[3,4-d]pyrimidines with anti-EGFR TK activity, have a low IC50 value against the BT474C cell line [45] (Figure 1). In 2018, our research group synthesized a series of 1H-pyrazolo[3,4-d]pyrimidine analogues as inhibitors of EGFRWT and EGFRT790M. Compound VI showed good inhibitory effects against the two types of EGFR besides a good apoptotic effect [46].

Rationale

The ATP-binding active pocket of the EGFR comprises five essential regions including the adenine and ribose binding pockets, in addition to the phosphate binding region, hydrophobic regions I and II. The ribose binding pocket and phosphate binding region are not essential for EGFR-TKIs intrinsic activity [47,48,49,50].
EGFR-TKIs have four common pharmacophoric features [51]: (i) A flat hetero aromatic system, terminal hydrophobic head, NH spacer, and hydrophobic tail. These moieties occupy and interact with the adenine binding pocket [52], the hydrophobic region I [51], linker region [53], and hydrophobic region II [47,54], respectively.
As shown in Figure 1, different moieties can occupy the adenine binding pocket as quinazoline (erlotinib I), quinoline (neratinib II), thieno[3,2-d]pyrimidine (olmutinib III), 1H-pyrazolo[3,4-d]pyrimidine (compounds IV, V, VI).
In continuation of our efforts in the discovery of EGFR-2 inhibitors [46,55,56,57], we designed and synthesized a new theobromine derivative as a potential compound that may exert a marked EGFR inhibitory activity and consequently inhibit the growth of tumor cells. The designed compound is a modified analog of 1H-pyrazolo[3,4-d]pyrimidine derivatives.
From a drug design point of view, the theobromine moiety was utilized in the current work to engage the adenine binding pocket. This moiety has six hydrogen acceptor atoms that can bind efficiently with the essential amino acid at the adenine binding pocket. In addition, the acetamide moiety was used as a linker. Furthermore, the hydrophobic head is to be inserted in the hydrophobic region I. Lastly, one of the two methyl groups at the 3- and 7-positions of theobromine moiety was used proposed to occupy the hydrophobic region II (Figure 1). The binding mode of the synthesized compound confirmed the design as each feature occupied its target pocket in the ATP binding site.

2. Results and Discussion

2.1. In Silico Studies

2.1.1. Molecular Docking against Wild and Mutant EGFR

The molecular modeling tool allowed medicinal chemists to envisage interactions between compounds and their biological target [58,59,60]. The designed theobromine derivative’s binding mode against wild (EGFRWT; PDB: 4HJO) and mutant (EGFRT790M; PDB: 3W2O) types of EGFR-TK, [61,62]. The co-crystallized ligands, erlotinib, and TAK-285, of wild and mutant types, respectively, were utilized as references.
The re-docking validation step of the co-crystallized ligands (erlotinib and TAK-285) showed acceptable root–mean–square deviation, RMSD, values of (1.40 and 1.10 Å, respectively), as presented in Figure 2 and Figure 3.
Erlotinib’s binding interactions with EGFRWT revealed that it occupied the major pockets (affinity value of −20.35 kcal/mol). A key hydrogen bond (H-B) with Met769, besides four hydrophobic interactions (H-I) with Leu694, Ala719, and Leu820, was observed in the adenine pocket. This was achieved via the quinazoline moiety. Moreover, the hydrophobic pocket I was occupied by the ethynylphenyl moiety through a network of H-Is with Ala719 and Val702, and Lys721 (Figure 4).
The designed theobromine derivative gave a comparable affinity value to erlotinib (−20.11 kcal/mol). In addition, it took the same orientation and interacted with EGFRWT active site similar to erlotinib. The 7-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purine of the designed compound was oriented into the adenine pocket of the EGFRWT with a H-B against the essential amino acid, Met769, and seven H-Is with Cys733, Leu820, Leu694, Leu786, Ala719, and Val 702. The NH group of the acetamide moiety was incorporated in an electrostatic attraction with Thr830. On the other side, the benzyl moiety occupied the hydrophobic pocket I via two H-Is with Leu764 and Lys721. The methyl group at the 4-position of xanthine moiety was oriented into the hydrophobic pocket II with two H-Is against Leu694 and Val 702 (Figure 5).
The docking outputs of the mutant EGFR (EGFRT790M) were investigated to support the docking results of the wild type (EGFRWT). Figure 6 explains the binding of TAK-285, the co-crystallized ligand, (TAK-285) to the EGFRT790M active site. The obtained findings and the reported data were identical [55].
As displayed in Figure 7, the designed theobromine derivative was bound to the catalytic site of the EGFRT790M in similar to TAK-285. The 7-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purine moiety was successfully buried in the adenine pocket to form one H-B with Met793 and four p-p bonds with Leu844, Leu718, Ala743, and Met793. Moreover, the terminal benzyl moiety interacted with the hydrophobic pocket I, forming an electrostatic interaction with Lys745. The methyl group at the 4-position of xanthine moiety was oriented into the hydrophobic pocket II, forming three H-Is with Leu844, Ala743, and Met793.

2.1.2. MD Simulations

The MD analyses carried out on the production run showed that, overall, the designed theobromine derivative-EGFR system was stable. The RMSD plot (Figure 8A) showed a stable average after the first 8 ns at 1.93 Å for the EGFR, blue curve, and the obtained complex, green curve. Furthermore, the RMSD of the designed theobromine derivative, red curve, showed a variation in values ranging from 0.5 Å to, approximately, 3.0 Å. The RMSD of the designed theobromine derivative can be divided into three parts. During the first 20 ns, it shows a large variation (from 0.5 to 2.72 Å) with an average of 1.41 Å, while the next 40 ns shows a small variation of approximately 1 Å (from 1.02 to 2.24 Å) with approximately the same average value of 1.41 Å. The last 40 ns show a similar variation in the values ranging from 1.05 to 3.19 Å with an average value of 1.96 Å. The radius of gyration, RoG, (Figure 8B), solvent accessible surface area, SASA, (Figure 8C), and H-bonds (Figure 8D) showed a stable protein (EGFR) fluctuation expressing an average of 19.58 Å, 15288 Å2, and 60 bonds, respectively. The fluctuated amino acids depicted in the root–mean–square fluctuation, RMSF, plot (Figure 8E) showed low fluctuation (less than 2 Å), with an exception of the free N-terminal reaching 6 Å and the loop region E841:V852. During the 100 ns, the designed theobromine derivative remained almost in the same place relating the EGFR center of mass (Figure 8F), with a 10.26 Å average.

2.1.3. MM-GBSA

The binding exact free energy investigation through molecular mechanics with generalized born and surface area, MM-GBSA, (Figure 9) indicates the different energy components that contribute to the process of binding. The designed theobromine derivative showed a total binding energy with the average value of −30.72 kcal/mol. The most favorable calculated contribution in the energy was for the Van Der Waals interaction (average value = −44.45 kcal/mol). Coming next, the electrostatic interactions (average of −25.26 kcal/mol). Furthermore, a decomposition analysis (Figure 10) was performed to disclose which amino acid residues within 1 nm of the designed theobromine derivative have more contribution to the binding. Leu694 (−1.1), Val702 (1.51), Ala719 (−1.02), Leu753 (−1.0), Thr766 (−1.13), Leu820 (−1.45), and Thr830 (−1.6) are the key amino acid residues that have a significant contribution of a value that is less than −1 kcal/mol.

2.1.4. Protein–Ligand Interaction Profiler (PLIP) Studies

Next, the trajectories of the computed MD were clustered, providing several representative frames that represent every obtained cluster. For every cluster, the PLIP webserver was employed to identify the number as well as the types of the interactions that occur between the designed theobromine derivative and EGFR. Table 1 denotes both number as well as types of interactions that produced from the PLIP webserver. In the first cluster representative, the predominated interaction is a H-B with three bonds. On the other hand, the two remaining cluster representatives had H-Is greater than the H-Bs. Besides obtaining types and numbers of interactions, the PLIP also generated a.pse file to see the 3D conformation of the designed theobromine derivative and its interaction with EGFR (Figure 11).

2.1.5. DFT

Geometry Optimization

All calculations were performed at the B3LYP/6-311G + +(d,p) level of theory. The full optimization of the compound was run without any constraints and presented in Figure 12. The bonding between the intermediate and the reactant through the C14-N2 bond is highlighted with the two angles on either side of the bond as shown in Figure 12. The polarity of any system is determined by the dipole moment (µ), which measures the interaction inside the molecule. The µ value of the title compound is 5.8158 Debye (D), and the calculated ground total energy (TE) is −30469.8 eV (Table 2).

Frontier Molecular Orbital (FMO) Analysis

From the FMO analysis, the calculated energy gap (Egap) between the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) for the ligand is −5.095 eV and the schematic diagram is presented in Figure 13. The Egap value is relatively small and reflects a narrow frontier orbital HOMO-LUMO gap. Based on the Dm and Egap values, the molecule is highly polarizable and chemically reactive [63]. The calculated energy of HOMO (EHOMO), LUMO (ELUMO), and Egap was listed in Table 2.

Global Reactive Indices and Total Density of State (TDOS)

The quantum calculations can be used effectively to obtain the global reactivity indices, which provides details about the chemical reactivity or inhibition ability of a molecule. Global reactive indices such as global chemical hardness (η) and softness (σ) describe the inhibitory ability of the molecule to be stable or reactive. A molecule is thought to be a good inhibitor when its hardness value (η) is small and softness value (σ) is high. All electronic parameters and reactivity indices of the molecule, such as ionization potential (IP), electrophilicity (ω), maximal charge acceptance (∆Nmax), chemical potential (µ), energy change (∆E), global chemical softness (σ), global chemical hardness (η), electronegativity (χ), and electron affinity (EA), were estimated according to Koopmans’ theory (more information in the supporting data).
Based on the calculated values of global reactive indices (Table 2), the molecule under investigation is chemically reactive and can be a good inhibitor against the EGFR protein [64].
At the border area, LUMO and HOMO may not provide a meaningful description of FMO due to the potential of quasi-degenerate levels. At equilibrium state, the product of the density of states of a chemical system and its probability distribution function gives the number of occupied states forunit volume. Such a number is utilized to study the physical properties of chemical systems. In this study, the TDOS analysis has been performed and the relative results confirmed that the compound under study had a significant small energy gap, and the highest intensity was reported for orbitals over LUMO orbital (Figure 14). Such results emphasized the promising inhibitor’s efficiency.

Surface Potential Mapping

The potential (electron + nuclei) mapping displays the distribution, molecular shape, size, and dipole moments of the electrostatic potential of the molecule [65]. Graphically, molecular electrostatic potential is shown in Figure 15, where the color-coded red, yellow, blue, and green regions denote the electron-rich, slightly electron-rich, electron-deficient, and neutral zones, respectively. The areas around the carbonyl groups represent the most negative potential zones and the slightly electron-rich regions with yellow colors are localized over terminal phenyl and N9 of purine moiety. The electron-deficient areas of blue color are localized on the purine substituted methyl (C13) and C8H25 of purine.

2.1.6. Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Profiling Study

Compounds are approved for use as drugs based on their pharmacokinetic evaluation along with their biological activity, so the pharmacokinetic evaluation of any new compound should be carried out at an early stage of its creation to help prevent the withdrawal of a drug after its approval. ADMET identifies absorption, distribution, metabolism, excretion, and toxicity, but despite the fact that in vitro studies can illustrate these properties, in silico analyses are more advantageous because of the limitations of cost, time, and effort, as well as the strict regulations regarding animal lives [66]. Computed ADMET parameters for the designed theobromine derivative against erlotinib as a reference molecule were determined using Discovery studio 4.0. According to ADMET results, the designed theobromine derivative and erlotinib exhibited similar good levels of solubility and intestinal absorption. Additionally, affinity to pass the blood–brain barrier, BBB, and to inhibit the cytochrome P450 (CYP2D6) was predicted as low and non-inhibitors, respectively. The two compounds (the designed theobromine derivative and erlotinib) showed a difference in the ability to bind with plasma protein as it was predicted to be less and more than 90%, respectively (Figure 16).

2.1.7. In Silico Toxicity Studies

In silico approaches to toxicity prediction have played an indispensable role in drug development, as it avoids ethical regulation, resource availability, and time-wasting in traditional in vitro and in vivo studies [67]. This approach has played an indispensable role in the discovery of new drugs and treatments [68]. In silico toxicity prediction uses structure–activity relationships (SARs) to predict toxicities, and in detail, the computer compares the chemical structural properties of the molecules to thousands of compounds that have been reported to be safe or toxic [69]. A toxicity model built in Discovery studio software was used to estimate eight toxicity parameters. Table 3 shows that the designed theobromine derivative demonstrated high levels of safety in the computed models.

2.2. Chemistry

Because the designed theobromine derivative demonstrated high affinity to bind with EGFR enzyme through different computational studies and it showed a good range of drug-likeness, it was semi-synthesized by the modification of theobromine. In the present work, potassium salt formation was achieved on the imidic nitrogen of theobromine VII [70] by treating it with alcoholic KOH while continuously stirring. The formed salt VIII was then reacted with N-benzyl-2-chloroacetamide X, which was formed via the reaction of phenylmethanamine IX with chloroacetylchloride in DMF, to produce the corresponding target product XI (Scheme 1).
The IR spectrum of compound XI was characterized by the appearance of carbonyl absorption bands at 1711 and 1656 cm−1. Moreover, the 1H NMR revealed the presence of amidic proton at 8.61 ppm. Moreover, a characteristic singlet signal appeared at 4.52 ppm corresponding to the CH2 group. The 13C NMR spectrum, which displayed distinct peaks at approximately 43.44, 42.52, 33.67, and 29.91 ppm corresponding to the two CH3 and two CH2 groups, respectively, further confirmed the validity of the proposed structure.
The chemical structure of compound XI (Figure 17) was confirmed through spectroscopic analysis. In detail, the NH proton resonated clearly at δ 8.61 as a triplet signal (J = 5.9 Hz) with the integration of one due to the presence of neighboring CH2. Two CH2 groups were detected at δ 4.30 (d, J = 5.9 Hz, 2H), which is adjacent to the NH, and at 4.52 (s, 2H) which is adjacent to the carbonyl. The proton of C-8, the only proton of the theobromine rings, resonated at δ 8.07 (s, 1H). The two methyls of the theobromine moiety (3, 7) resonated as two singlet peaks at δ 3.44, and 3.90, respectively. Furthermore, a pattern of monosubstituted benzene ring was discovered at δ 7.37–7.31 (2H) and 7.26 (3H). Similarly, the 13C NMR showed three carbonyls at δ 167.50, for the acetamide moiety, in addition to the two up-field carbonyls of the theobromine moiety because of the high anisotropic effects at δ 154.73 and 151.39. Additionally, C-4, C-5, and C-8 of the theobromine moiety resonated at 148.97, 107.18, and 143.53, respectively. The monosubstituted benzene ring shoed its characteristic signals at 139.72, 128.75 (2C), 127.57 (2C), and 127.25. Two methylene and two methyl groups were detected at 43.44, 42.52, 33.67, and 29.91, respectively.

2.3. Biological Evaluation

2.3.1. EGFR Inhibition

To examine the design and the computational results that indicated the strong binding affinity of compound XI to the EGFR enzyme, compound XI was tested in vitro against the EGFR enzyme in comparison with erlotinib. As shown in Figure 18, compound XI strongly inhibited the EGFR enzyme with 17.2 nM that was bordering erlotinib’s value. The obtained results were consistent with the acquired in silico results and confirmed the strong suppressing potential of compound XI.

2.3.2. Cytotoxicity and Safety

An in vitro cytotoxicity assay to evaluate compound XI’s EGFR inhibition against cancer was conducted using the A549 and HCT-116 malignant cell lines against erlotinib as a reference. Compound XI demonstrated IC50 values of 21.99 and 22.02 µM, respectively (Table 4). The anticancer potentials of compound XI were very close to those of erlotinib (6.73 and 16.35 µM, respectively).
The cytotoxic potential of compound XI against the W138 normal human cell line was evaluated to confirm its in silico safety results and determine its selectivity against cancer cell lines. Compound XI displayed excellent safety results, expressing a high IC50 value of 49.44 µM (safer than erlotinib) and very high selectivity indexes (SI) against both examined cell lines of 2.2 (Figure 19).

2.3.3. Cell Cycle Analysis and Apoptosis Assay

Initially, flow cytometric analysis of cell cycle phases was carried out [71,72]. Compound XI was treated with A549 cells for 72 h at a concentration of 21.99 µM. Then, the different stages of the cell cycle were examined. Compound XI decreased the growth of cells in the Sub-G1 and G1 phases from 0.87% to 0.80% and from 43.47% to 16%, respectively. Conversely, the f A549 population percentage was significantly increased from 12.10 for control cells to 19.84 for XI-treated cells in the G2/M phase (Table 5 and Figure 20). To confirm the apoptotic effects of XI, A549 cells were stained with Annexin V and PI double stains after treatment of with 21.99 µM of XI for 72 h [73,74]. Comparing the control, compound XI induced a higher number of apoptotic cells. Compound XI caused a significant increase in the apoptotic cells percentage in both early apoptosis (from 0.05% to 24.02%) and late apoptosis (from 0.49% to 41.70%). A noticeable change in the total apoptosis percentage by 65.72 was observed, compared to 0.54% in the control cells (Figure 20 and Table 6). In conclusion, compound XI primarily arrested the cancer cell cycle at the G2/M stage caused cytotoxic activities that may be due to programmed apoptosis. (See Supplementary Materials).

3. Experimental

3.1. In Silico Studies

3.1.1. Docking Studies

The molecular docking was carried out by MOE2014 software [75]. Supplementary data give a detailed explanation.

3.1.2. MD Simulations

CHARMM-GUI web server and GROMACS 2021 were utilized as an MD engine [76,77]. Supplementary data give a detailed explanation.

3.1.3. MM-GBSA

The Gmx_MMPBSA package was utilized [78]. Supplementary data give a detailed explanation.

3.1.4. DFT

Gaussian 09 and GaussSum3.0 programs [79] were utilized [80]. Supplementary data give a detailed explanation.

3.1.5. ADMET Studies

ADMET profile was carried out by Discovery Studio 4.0 [81]. Supplementary data give a detailed explanation.

3.1.6. Toxicity Studies

The toxicity profile was carried out by Discovery Studio 4.0. Supplementary data give a detailed explanation.

3.2. Chemistry

Synthesis of Compound XI

To a solution of the potassium salt of theobromine VIII (0.001 mol, 0.25 g) in dry DMF (10 mL), N-benzyl-2-chloroacetamide X (0.001 mol, 0.21 g) was added and the mixture was heated for 5 h. The mixture was then cooled and the produced precipitate was filtered, washed with water, and crystallized from ethanol to attain the final target compound XI (Scheme 2).
Off-white crystal (yield, 84%); m. p. = 257–259 °C; IR (KBr) ν cm−1: 3282, 3108 (NH), 2929 (CH aliphatic), 1711, 1656 (C = O); 1H NMR (400 MHz, DMSO-d6) δ 8.61 (t, J = 5.9 Hz, 1H), 8.07 (s, 1H), 7.37–7.31 (m, 2H), 7.26 (d, J = 7.5 Hz, 3H), 4.52 (s, 2H), 4.30 (d, J = 5.9 Hz, 2H), 3.90 (s, 3H), 3.44 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 167.50, 154.73, 151.39, 148.97, 143.53, 139.72, 128.75 (2C), 127.57 (2C), 127.25, 107.18, 43.44, 42.52, 33.67, 29.91. For C16H17N5O3 (327.34).

3.3. Biological Studies

3.3.1. In Vitro Egfr Inhibition

This was performed using the Human EGFR ELISA kit. The supplementary data provide a thorough explanation.

3.3.2. In Vitro Antiproliferative Activity

MTT procedure was utilized. The supplementary data provide a detailed explanation.

3.3.3. Safety Assay

The normal cell lines, W138, were utilized. The supplementary data provide a detailed explanation.

3.3.4. Cell Cycle Analysis and Apoptosis

The effect of compound XI on cell cycle distribution and apoptosis was performed using flowcytometry analysis technique. The supplementary data provide a detailed explanation.

4. Conclusions

Based on the essential structural properties of EGFR inhibitors, a new theobromine derivative was designed as an inhibitor. The potentiality of the designed theobromine derivative against EGFR was demonstrated by molecular docking against both wild and mutant types. The binding was confirmed by six MD (over 100 ns), two MM-GBSA, PLP, and four DFT experiments. In addition, an ADMET analysis confirmed the general likeness and safety. In vitro results were consistent with the in silico results, displaying EGFR inhibition with an IC50 value of 17.23 nM and cytotoxic properties against A549 and HCT-116 cell lines with IC50 values of 21.99 and 22.02 µM, respectively. Compound XI was much safer against the healthy W138 cell line (IC50 = 49.44 µM) than erlotinib (IC50 = 31.17 µM), showing a selectivity index of 2.2. Compound XI arrested the A549 cell cycle at the G2/M stage and induced apoptosis. According to these results, compound XI could be a new lead compound in the discovery of anti-EGFR candidates through further modifications and more examinations.
The results show that compound XI (the theobromine derivative) is a lead compound that could be employed for further modifications or in vivo and preclinical studies.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/molecules27185859/s1. Detailed toxicity report, the detailed methods, spectral data, and toxicity report.

Author Contributions

Formal analysis, A.M.M., A.A.A.; Funding acquisition, E.B.E.; Methodology, R.G.Y., H.E., D.Z.H. and I.M.I.; Project administration, A.M.M.; Supervision, I.H.E.; Validation, I.H.E.; Writing—original draft, H.E.; Writing—review & editing, A.M.M. and I.H.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2022R116), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors extend their appreciation to the Research Center at AlMaarefa University for funding this work.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Compound XI is available from the authors.

References

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Molecules 27 05859 g001 550
Figure 1. The design rationale of the targeted compound.
Figure 1. The design rationale of the targeted compound.
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Molecules 27 05859 g002 550
Figure 2. Validation step of erlotinib.
Figure 2. Validation step of erlotinib.
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Molecules 27 05859 g003 550
Figure 3. Validation step of TAK-285.
Figure 3. Validation step of TAK-285.
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Molecules 27 05859 g004a 550Molecules 27 05859 g004b 550
Figure 4. 3D and 2D binding mode of erlotinib into the active site of EGFRWT.
Figure 4. 3D and 2D binding mode of erlotinib into the active site of EGFRWT.
Molecules 27 05859 g004aMolecules 27 05859 g004b
Molecules 27 05859 g005a 550Molecules 27 05859 g005b 550
Figure 5. MS, 3D, and 2D binding pattern of the designed theobromine derivative with the active site of EGFRWT.
Figure 5. MS, 3D, and 2D binding pattern of the designed theobromine derivative with the active site of EGFRWT.
Molecules 27 05859 g005aMolecules 27 05859 g005b
Molecules 27 05859 g006 550
Figure 6. TAK-285 docked into EGFRT790M active site.
Figure 6. TAK-285 docked into EGFRT790M active site.
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Molecules 27 05859 g007 550
Figure 7. Binding of the designed theobromine derivative with EGFRT790M.
Figure 7. Binding of the designed theobromine derivative with EGFRT790M.
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Molecules 27 05859 g008a 550Molecules 27 05859 g008b 550Molecules 27 05859 g008c 550
Figure 8. Different measurements produced from the trajectory. (A) RMSD: EGFR is blue, the designed theobromine derivative is red, and the complex is green c, (B) RoG, (C) SASA, (D) H-bonds number of, (E) RMSF, and (F) mass distance center between the designed theobromine derivative and the EGFR.
Figure 8. Different measurements produced from the trajectory. (A) RMSD: EGFR is blue, the designed theobromine derivative is red, and the complex is green c, (B) RoG, (C) SASA, (D) H-bonds number of, (E) RMSF, and (F) mass distance center between the designed theobromine derivative and the EGFR.
Molecules 27 05859 g008aMolecules 27 05859 g008bMolecules 27 05859 g008c
Molecules 27 05859 g009 550
Figure 9. Different energetic components of MM-GBSA and their average values. Bars represent the standard deviation.
Figure 9. Different energetic components of MM-GBSA and their average values. Bars represent the standard deviation.
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Molecules 27 05859 g010 550
Figure 10. Binding-free energy decomposition of the EGFR, the designed theobromine derivative complex.
Figure 10. Binding-free energy decomposition of the EGFR, the designed theobromine derivative complex.
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Molecules 27 05859 g011a 550Molecules 27 05859 g011b 550
Figure 11. (C1–C3) Types and number of different interactions produced from PLIP for each cluster representative. H-bond: Blue solid line, H-I: dashed gray line, amino acids: blue sticks representation, and the designed theobromine derivative: orange sticks representation.
Figure 11. (C1–C3) Types and number of different interactions produced from PLIP for each cluster representative. H-bond: Blue solid line, H-I: dashed gray line, amino acids: blue sticks representation, and the designed theobromine derivative: orange sticks representation.
Molecules 27 05859 g011aMolecules 27 05859 g011b
Molecules 27 05859 g012 550
Figure 12. The optimized chemical structure of the compound at B3LYP/6-311G + +(d,p) level.
Figure 12. The optimized chemical structure of the compound at B3LYP/6-311G + +(d,p) level.
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Molecules 27 05859 g013 550
Figure 13. The FMO analysis; HOMO and LUMO at the ground state of the molecule at B3LYB/6-311 + +G(d,p).
Figure 13. The FMO analysis; HOMO and LUMO at the ground state of the molecule at B3LYB/6-311 + +G(d,p).
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Molecules 27 05859 g014 550
Figure 14. Molecular frontier orbital spectrum, total density of states (TDOS) and energies obtained at B3LYP/6-311 + +G(d,p) level of the designed compound.
Figure 14. Molecular frontier orbital spectrum, total density of states (TDOS) and energies obtained at B3LYP/6-311 + +G(d,p) level of the designed compound.
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Molecules 27 05859 g015 550
Figure 15. Molecular electrostatic potential map of the designed derivative at the 6-311G + +(d,p) basis set.
Figure 15. Molecular electrostatic potential map of the designed derivative at the 6-311G + +(d,p) basis set.
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Molecules 27 05859 g016 550
Figure 16. Computational prediction of ADMET parameters for the designed theobromine derivative and erlotinib.
Figure 16. Computational prediction of ADMET parameters for the designed theobromine derivative and erlotinib.
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Molecules 27 05859 sch001 550
Scheme 1. Synthesis of the target theobromine derivative XI.
Scheme 1. Synthesis of the target theobromine derivative XI.
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Figure 17. Chemical structure of compound XI.
Figure 17. Chemical structure of compound XI.
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Molecules 27 05859 g018a 550Molecules 27 05859 g018b 550
Figure 18. In vitro EGFR inhibition of compound XI (A) and erlotinib (B).
Figure 18. In vitro EGFR inhibition of compound XI (A) and erlotinib (B).
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Molecules 27 05859 g019 550
Figure 19. In vitro anti-proliferative activities of the compound XI and erlotinib against EGFR, cancer and normal cell lines.
Figure 19. In vitro anti-proliferative activities of the compound XI and erlotinib against EGFR, cancer and normal cell lines.
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Molecules 27 05859 g020 550
Figure 20. (A) Cell cycle and (B) apoptotic analysis of compound XI and control untreated A549 cell line.
Figure 20. (A) Cell cycle and (B) apoptotic analysis of compound XI and control untreated A549 cell line.
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Molecules 27 05859 sch002 550
Scheme 2. N-Benzyl-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetamide (XI).
Scheme 2. N-Benzyl-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetamide (XI).
Molecules 27 05859 sch002
Table
Table 1. Interactions (number and types) detected from the PLIP webserver. Bold amino acids are common in all clusters.
Table 1. Interactions (number and types) detected from the PLIP webserver. Bold amino acids are common in all clusters.
Cluster NumberH-IsAmino Acids in EGFRH-BsAmino Acids in EGFR
C11F8323Lys721-Thr830-Asp831
C24Leu764-Thr766-Phe832-Leu8342Lys721-Asp831
C33Leu764-Phe832-Leu8342Lys721-Thr830
Table
Table 2. The calculated global reactivity indices and energetic parameters for the selected compound.
Table 2. The calculated global reactivity indices and energetic parameters for the selected compound.
IPEAμ (eV)χ (eV)η (eV)σ (eV)ω (eV)µ (D)TE (eV)∆Nmax∆E (eV)
−1.512−6.607−4.0594.059−2.548−0.393−20.9875.816−30469.8−1.59320.987
Table
Table 3. In silico toxicity studies of the designed theobromine derivative and erlotinib.
Table 3. In silico toxicity studies of the designed theobromine derivative and erlotinib.
Comp.FDA Rodent Carcinogenicity
(Mouse-Male)		Carcinogenic Potency TD50
(Mouse) a		Ames MutagenicityRat Maximum Tolerated Dose
(Feed) b		Rat Oral LD50 bRat Chronic LOAEL bSkin IrritancyOcular Irritancy
The designed compoundNon-Carcinogen70.942Non-Mutagen0.0241.5750.039Non-IrritantMild
Erlotinib39.7710.0830.6620.036
a Unit: mg/kg/day. b Unit: g/kg.
Table
Table 4. In vitro anti-proliferative activities of the compound XI and erlotinib against EGFR, A549 HCT-116, and WI-38 cell lines.
Table 4. In vitro anti-proliferative activities of the compound XI and erlotinib against EGFR, A549 HCT-116, and WI-38 cell lines.
Comp.In Vitro Cytotoxicity IC50 (µM) aA549
(SI)		HCT-116 (SI)EGFR, IC50 (nM)
A549HCT-116WI-38
Compound XI21.9922.0249.442.22.217.23
Erlotinib6.7316.3531.174.61.95.91
a Data are presented as the mean of the IC50 values from three different experiments.
Table
Table 5. Cell cycle analysis of compound XI and control untreated A549 cell line.
Table 5. Cell cycle analysis of compound XI and control untreated A549 cell line.
SampleCell Cycle Distribution (%) a
% Sub-G1% G1% S% G2/M
A5490.87 ± 0.3543.47 ± 3.9943.56 ± 2.3112.10 ± 2.04
Compound XI/A5490.80 ± 0.1716.01 ± 5.0063.35 ± 2.50 *19.84 ± 2.69
a All values are calculated as the mean of triplicates. * p < 0.1.
Table
Table 6. Effect of compound XI on A549 cells’ stages of death.
Table 6. Effect of compound XI on A549 cells’ stages of death.
SampleViable a
(Left Bottom)		Apoptosis aNecrosis a
(Left Top)
Early
(Right Bottom)		Late
(Right Top)
A54999.44 ± 0.460.05 ± 0.020.49 ± 0.460.02 ± 0.01
Compound XI/A54932.55 ± 2.9824.02 ± 1.52 **41.70 ± 1.76 **1.73 ± 0.30
a All values are calculated as the mean of at least three different experiments. ** p < 0.01.
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MDPI and ACS Style

Elkaeed, E.B.; Yousef, R.G.; Elkady, H.; Alsfouk, A.A.; Husein, D.Z.; Ibrahim, I.M.; Metwaly, A.M.; Eissa, I.H. New Anticancer Theobromine Derivative Targeting EGFRWT and EGFRT790M: Design, Semi-Synthesis, In Silico, and In Vitro Anticancer Studies. Molecules 2022, 27, 5859. https://doi.org/10.3390/molecules27185859

AMA Style

Elkaeed EB, Yousef RG, Elkady H, Alsfouk AA, Husein DZ, Ibrahim IM, Metwaly AM, Eissa IH. New Anticancer Theobromine Derivative Targeting EGFRWT and EGFRT790M: Design, Semi-Synthesis, In Silico, and In Vitro Anticancer Studies. Molecules. 2022; 27(18):5859. https://doi.org/10.3390/molecules27185859

Chicago/Turabian Style

Elkaeed, Eslam B., Reda G. Yousef, Hazem Elkady, Aisha A. Alsfouk, Dalal Z. Husein, Ibrahim M. Ibrahim, Ahmed M. Metwaly, and Ibrahim H. Eissa. 2022. "New Anticancer Theobromine Derivative Targeting EGFRWT and EGFRT790M: Design, Semi-Synthesis, In Silico, and In Vitro Anticancer Studies" Molecules 27, no. 18: 5859. https://doi.org/10.3390/molecules27185859

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