Semi-synthesized anticancer theobromine derivatives targeting VEGFR-2: in silico and in vitro evaluations

Abstract Vascular endothelial cell proliferation and angiogenesis are all crucially impacted by Endothelial Growth Factor Receptor-2 (VEGFR-2). Its expression is significantly boosted throughout pathologic angiogenesis causing the development of tumors. Sothat, inhibition of VEGFR-2 has crucial role in cancer treatment. In this study, novel semisynthetic theobromine derivatives were rationally designed as VEGFR-2 inhibitors and subjected to in vitro testing for their ability to block VEGFR-2 activation. Furthermore, the antiproliferative effects of these derivatives were evaluated. Compound 7 g exhibited the most potent anti-VEGFR-2 activity, with an IC50 value of 0.072 µM, and demonstrated excellent dose-dependent inhibitory activity against both MCF-7 and HepG2 cancer cells with IC50 values of 19.35 and 27.89 µM, respectively. Notably, compound 7 g exhibited high selectivity indices of 2.6 and 1.8 against MCF-7 and HepG2 cells, respectively. Compound 7 g induced G2/M phase cell cycle arrest, promoted apoptosis, and boosted immunomodulation by downregulating TNF-α expression and upregulating IL-2 levels in MCF-7 cells. The molecular docking analysis revealed that compound 7 g could bind effectively to the active site of VEGFR-2, and molecular dynamic simulations confirmed the stability of the VEGFR-2/compound 7 g complex. Furthermore, ADME and toxicity profiling indicated the potential suitability of these compounds as drug candidates. In summary, compound 7 g hold promise as a VEGFR-2 inhibitor. Communicated by Ramaswamy H. Sarma


Introduction
Despite the discovery of a vast array of anticancer drugs, the incidence and prevalence of cancer continue to increase considerably (Bray et al., 2018).Discovery of targeted chemotherapeutic agents that can effectively interact with specific molecular targets of cancer cells and inhibit their growth remains a significant challenge for researchers (Ward et al., 2021).Such a task is multifaceted and necessitates a comprehensive understanding of the intricate molecular mechanisms underlying cancer to develop precise interventions that can selectively target cancer cells while minimizing harm to healthy tissues (DeVita & Chu, 2008;Fidler & Ellis, 2000).
One of the most targeted proteins for treating cancer is the vascular endothelial growth factor receptor-2 (VEGFR-2).VEGFR-2 is a type of tyrosine kinase receptor that plays an essential role in various cellular processes, including cell proliferation, adhesion, division, and angiogenesis (Shah et al., 2021).Studies have demonstrated that VEGFR-2 receptors in cancer cells are more active than those in normal cells, making it possible to develop a drug that specifically targets angiogenesis in tumor cells without affecting normal cells (Elrazaz et al., 2021).
Upon activation of VEGFR-2 by its ligand, VEGF, it encourages the development of neighboring vessels to assist the transport of nutrients, oxygen, and growth factors needed for cancer growth and metastasis (Simons et al., 2016).VEGF/VEGFR-2 facilitated angiogenesis supports violent natures and leads to a high mortality rate of cancer (Lian et al., 2019) (the 2nd leading cause of death worldwide) (WHO, 2023).VEGFR2 was confirmed to be present in cancer cells in addition to its constitutive expression on endothelial cells.VEGFR-2 is overexpressed in many types of tumors as lung cancer (Riquelme et al., 2014), breast cancer (Zhang et al., 2017) renal cell carcinoma, hepatocellular carcinoma (Pengcheng et al., 2017), gastrointestinal cancer (Mahfouz et al., 2017), glioblastoma (Lu-Emerson et al., 2015), bladder cancer (Zhong et al., 2017), ovarian cancer (Jang et al., 2017), and osteosarcoma (Liu et al., 2017).
Depending on the chemical structures of the reported VEGFR-2 inhibitors, it was found that VEGFR-2 inhibitors have four main pharmacophoric features which are essential for maximal fitting in the active site of VEGFR-2.As shown in Figure 2, these features are (i) a heteroaromatic system that can bind the hinge region of the active site, (ii) a spacer moiety that can occupy the linker region, (iii) a pharmacophore group that consists of at least one hydrogen bond donor and one hydrogen bond acceptor moieties and can occupy DFG motif region, and (iv) a hydrophobic tail that can occupy the allosteric pocket of the ATP binding domain (Lee et al., 2010;Wang et al., 2013).
Compounds IX, X, and XI were selected as lead structures for chemical modifications.The modification steps comprised the replacement of the 3-methylquinoxaline moiety with a methylxanthine moiety which was designed to occupy the hinge region of the active site.This modification was carried out to scavenge the advantages of the methylxanthine moiety.It has many hydrophilic groups that can form several hydrogen bonds in the active site with an increased level of aqueous solubility.Additionally, the N-phenylacetamide and amide moieties were not changed in the target compounds.On the other hand, many substituted aromatic groups were used as hydrophobic tails in the target compounds (Figure 3).

Chemistry
The target theobromine derivatives 7a-g were synthesized following the general synthetic route outlined in Scheme 1. Firstly, 4-aminobenzoic acid 1 was treated with chloroacetyl chloride in DMF to afford the chloroacetamide intermediate 2.Then, compound 2 was acylated using thionyl chloride to yield 4-(2-chloroacetamido)benzoyl chloride 3 as described in the reported methodologies (Alanazi et al., 2021b(Alanazi et al., , 2021c)).Treating of 3 with commercially available amines namely, 4-aminoacetophenone, 4-chloroaniline, 4-fluoroaniline, 4methoxyaniline, 4-methylaniline, 3-methoxyaniline, and 3methylaniline in acetonitrile in the presence of triethylamine (TEA), afforded the target intermediates 4a-g, respectively.On the other hand, theobromine 5 was heated with alcoholic KOH with continuous stirring to afford the corresponding potassium salt 6 (Taghour et al., 2022b).To finalize the target compounds 7a-g, the formed potassium salt 6 was heated with the previously prepared intermediates 4a-g in dry DMF using a catalytic amount of KI.
The spectral data supported the structures of the synthesized derivatives, where IR spectra exhibited characteristic bands at the ranges of 3272-3451 cm À 1 and 1638-1677 cm À 1 corresponding to NH and C ¼ O groups, respectively.While 1 H NMR analysis revealed the presence of singlet signals at a range of d 10.08-10.65 ppm corresponding to the NH amide groups.Also, it showed singlet signals at a range of d 4.40-4.72ppm and 8.08-8.10ppm corresponding to the aliphatic linker methylene protons and the methine proton of theobromine nucleus. 13C NMR spectra of the final derivatives showed a peak for a de-shielded methylene group.Also, it revealed the presence of down-field peaks for amide carbonyl moiety.The methylene group was confirmed by the presence of a singlet signal at d 4.43 ppm. 13C NMR spectrum showed the presence of two peaks at 34.51, and 18.41 corresponding to CH 2 and CH 3 groups, respectively.

In-vitro anticancer effects
Two human cancer cell lines (MCF-7; breast cancer and HepG2; liver carcinoma) were used to evaluate the antiproliferative activities of the synthesized compounds.MTT assay  (Mosmann, 1983) was applied in this test using sorafenib as a positive standard cytotoxic drug.MCF-7 and HepG2 were used in this test because VEGFR-2 is highly expressed in both breast cancer and hepatocellular carcinoma (Chu et al., 2013;Guo et al., 2003).The concentrations of the tested compounds that produced 50% growth inhibition of cancer cells (IC 50 in mM) are presented in Table 1.
The cytotoxic results showed that compound 7d was the most potent member exhibiting exceptionally very strong cytotoxic activity against MCF-7 and HepG2 with IC 50 5 4.32 ± 0.2 and 5.84 ± 0.3 mM, respectively, much less than that of sorafenib (IC 50 ¼ 7.26 ± 0.3 and 9.18 ± 0.6 mM).Also, compound 7 g displayed promising anticancer activity with IC 50 values of 19.35 ± 1.3and 27.89 ± 2.0 mM against MCF-7 and HepG2, respectively.In addition, derivatives 7b, c, e, and f showed moderate to weak activities against the aforementioned cancer cell lines with IC 50 values ranging from 24.78 ± 1.8 to 86.46 ± 4.0 mM.Finally, compound 7a appeared to be non-active against the tested two cell lines.
On analyzing the SAR of the synthesized compounds.Investigating the effect of the substitution at the 3-position with electron-donating groups, the derivative with a 3methyl substitution (7 g) is more active than that with a 3methoxy group (7f).For the substitution at the 4-position, compounds substituted with electron-withdrawing groups can be arranged in order of increasing activity as follows 4acetyl (7a) < 4-fluoro (7c) < 4-chloro (7b).The derivatives those substituted with electron-donating groups showed higher activities (7d, e) in which substitution with a 4methoxy group (compound 7d) provided the most active candidate in all derivatives.

In vitro VEGFR-2 enzyme assay inhibition
All the synthesized compounds were evaluated for their VEGFR-2 inhibitory activities utilizing sorafenib as a reference drug.The results were presented as IC 50 values (expressed in mM) in Table 1.Comparing to sorafenib (IC 50 ¼ 0.015 mM), compounds 7 g and 7d showed the highest VEGFR-2 inhibitory activities with IC 50 values of 0.072 and 0.301 mM, respectively.In addition, compounds 7a and 7f showed moderate activities with IC 50 values of 0.98 and 0.95 mM, respectively.On contrast, compounds 7b, 7c, and 7e showed weak activities with IC 50 values of 1.74, 1.83, and 1.57 mM, respectively.

Cytotoxicity against normal cells
One of the major drawbacks of chemotherapy is the inadvertent harm it can cause to healthy cells (Chabner & Roberts, 2005).Therefore, it is important to investigate whether the antiproliferative potential of a drug such as 7 g is due to its effect as an anticancer agent or simply its general toxicity.To this end, normal human lung fibroblasts, WI-38, cell lines were employed to assess the safety of 7 g.
Results demonstrate that 7 g exhibits low toxicity to WI-38 cells with an IC 50 of 51.14 ± 2.47 mM.The high safety margins of 7 g were indicated through the expression of high selectivity indices towards MCF-7 and HepG2 cell lines of 2.6 and The IC 50 values equal the mean ± SD of three different tests.
1.8, respectively.These values present 7 g as a lead compound that may have potential as an anticancer with reduced toxicity towards healthy cells.

Cell cycle test
Eukaryotic cells undergo a complex process of replication through successive cell cycles.Antiproliferation drugs are designed to affect specific phases of the cell cycle, inducing growth arrest at specific checkpoints and potentially triggering apoptosis (Pietenpol & Stewart, 2002).Accurate identification of the checkpoint targeted by the drug is essential for effective cancer treatment, as it will prevent the cancer cells from progressing through the cell cycle, ultimately leading to the inhibition of cell proliferation.
The study exposed MCF-7 cells to 19.35 mM of 7 g and observed changes in the cell cycle.Results showed that 7 g significantly increased the cell population at the G2/M phase compared to untreated cells from 16.41 to 24.89.In contrast, compound 7 g induced a decrease in the cell population at the G0-G1 phase (from 59.04 to 54.51%) and S phase (from 24.55 to 20.60%).These findings revealed the cell cycle arrest at the G2/M phase (Figure 4 and Table 2).

Apoptosis induction
To assess the apoptotic effect of 7 g (19.35 mM) on MCF-7 cells, the Annexin-V/propidium iodide staining (Annexin-V FTIC) test was conducted.The results (Supplementary data) showed a significant increase in early and late apoptosis in MCF-7 cells treated with 7 g (17.65 and 7.21%, respectively) compared to untreated cells (0.43 and 1.71%, respectively).These findings demonstrate the potent apoptotic effects of 7 g on the MCF-7 cells.

Quantitative RT-PCR assay
Bax and Bcl-2 are two vital genes controlling mitochondrial apoptosis.During the process of apoptosis, these genes have opposite functions.While Bcl-2 is anti-apoptotic, Bax promotes apoptosis (Callagy et al., 2008;McDonnell & Korsmeyer, 1991).Thus, measuring Bax and Bcl-2 is an essential factor to understand mitochondrial apoptosis.Through quantitative reverse transcription-polymerase chain reaction qRT-PCR.
The study found that compound 7 g at a concentration of 19.35 mM had a significant impact on MCF-7 cells.Specifically, it upregulated the expression of Bax by more than 4.5 times and downregulated Bcl-2 by more than 6 times (Table 3 and Figure 5).
At the same time, compound 7 g was further subjected to another qRT-PCR assay to evaluate its potential immunomodulatory effects on MCF-7 cells.Two immunity-related markers, specifically human tumor necrosis factor alpha (TNFa) and interleukin 2 (IL-2) were quantitatively assessed.A potent inhibitor, 5-fluorouracil (5FU), was employed as a positive control.The findings indicated that 7 g exhibited a strong suppressive effect on the TNF-a (2.5-fold) and stimulatory effect on the IL-2 (3.3-fold) compared to the control cells (Table 3 and Figure 5).

Docking study
It was an important part of our research to get the possible reasons for the activity of the designed members 7a-g.
Therefore, we docked these compounds into the VEGFR-2 crystal structure available in the Protein Data Bank (PDB: 2OH4) using MOE2019.Validation of the docking method has been proven by re-docking the native ligand against the active site.The results revealed that both native and docked ligands were superimposed pretty well (Figure 6).
The binding mode of sorafenib in the active site cavity of VEGFR-2 was compatible with the reported one (Alsaif et al., 2021).It binds the hinge region through its N-methyl picolinamide part forming two hydrogen bonds with the backbone amide NH and the carbonyl's oxygen of Cys917.The amide group formed three hydrogen bonds with the side chain of Glu883 and the backbone NH of Asp1044 in the DFG-motif region which is electron rich region.The linker   # : means significant P value from 5FU group, significant P value < 0.05 & by using One-way ANOVA followed by Tukey's post hoc multiple comparison tests.
region and the terminal hydrophobic regions were occupied by the central phenyl ring and the 4-chloro-3-(trifluoromethyl)benzene moiety, respectively (Figure 7).
The proposed binding modes of compounds 7a-g in the ATP binding pocket of VEGFR-2 were analyzed.As we expected in the rationale part, the purine fragment of the designed derivatives could occupy the hinge region lined with Cys917, Leu1033, Ala864, Leu838, and Phe916 residues.In addition, the linker region lined with Val914, Phe1045, and Lys866 residues was occupied by the central phenyl moiety.Just like sorafenib, the amide moiety of compounds 7a-g could also form two hydrogen bonds with the two critical residues Glu883 and Asp1044 in the DFG motif region.Interestingly, the presence of the different terminal aromatic fragments including 4-acetylphenyl (7a), 4-chlorophenyl (7b), 4-fluorophenyl (7c), 4-methoxyphenyl (7d), 4-methylphenyl (7e), 3-methoxyphenyl (7f), and 4-methylphenyl (7 g) allowed the designed inhibitors to fit very well with the allosteric pocket and interacted the hydrophobic backbone lined with Ile886, Ile890, and Leu1017.These results rationalized the potent VEGFR-2 inhibitory activities of the designed candidates (Figure 8).

MD simulation
The analysis of the production run showed that the conformation of 7 g inside the binding pocket of VEGFR-2 and its distance from the center of mass of the protein was stable throughout the time of the simulation (100 ns).The production run indicates a rising trend in RMSD plots for VEGFR-2 protein and VEGFR-2-7g complexe over the first 20 ns, before stabilizing at an average of 2.6 Å. (Blue and red lines in Figure 9A).While the 7g's RMSD (green line in Figure 9A) remains relatively constant at about 1.8 Å during the first 82 ns, then increases to an average of around 2.9 Å. Figure 9G shows that 7 g motion inside the VEGFR-2 binding pocket is responsible for this increase in RMSD. Figure 9B and C show two different average values for the radius of gyration (RoG) and the SASA, respectively.RoG and SASA show an average of around 20.6 Å and 17647 Å 2 over the first 56 ns, then decreasing slightly to 20.3 Å and 16903 Å 2 for the remainder of the simulation.The average number of H-bonds is 71 bonds, however, it varies over time as seen in Figure 9D.Overall, this indicates that the VEGFR-2 protein's structure is stable.The RMSF plot (Figure 9E) reveals that, except for the N-terminus (12.4 Å), the Lys1053:Asp1062 loop (4.8 Å), and the C-terminus (6.2 Å), amino acids show stable fluctuation (less than 2 Å).With an average distance of 8.2 Å between the centers of mass of 7 g and the VEGFR-2 protein, the binding is stable (Figure 9F).
Figure 12 shows the interactions between amino acids and the persistence of those interactions throughout the simulation, revealing that ten amino acids (Leu838, Val846, Ala864, Leu887, Val897, Val914, Leu1033, Cys1043, Asp1044, and Phe1045) had extremely persistent hydrophobic contacts (with at least 93% occurrence).Pi-stacking interaction with ligand established by Phe1045 is less stable (occurs 75% of the time).Only Cys917 and Asp1044 have an H-bonding frequency of at least 75%. Figure 13 shows the representative frames (4 frames) produced from clustering using TTClust and the types of interactions detected using the PLIP webserver.

In silico ADME analysis
The investigation of pharmacokinetic and biological potentialities is crucial in the approval of new medicines.So, it is vital to evaluate pharmacokinetic characteristics of any new chemical as early as possible in the drug discovery process to avoid delays in approval or even late withdrawal (Ferreira & Andricopulo, 2019).The ADME characteristics of the new semi-synthesized theobromine derivatives were computationally investigated using Discovery Studio 4.0, and sorafenib as a reference drug (as shown in Figure 14 and Table 4).Interestingly, the results of the computational analysis showed that all of the theobromine derivatives had a penetration of the blood-brain barrier (L_BBB) with very low levels and good levels of aqueous solubility (L_Sol).Most of the theobromine derivatives, including 7 g, exhibited good levels of intestinal absorption (L_Abs).Fortunately, all of the theobromine derivatives were predicted to be non-inhibitors against the cytochrome P4502D6 and non-hepatotoxic (Hep_ T).Also, all of the theobromine derivatives were predicted to bind the plasma protein binding (L_PPB) model at levels less than 90%.

Toxicity studies
Eight computational models were employed through the Discovery Studio software to estimate the toxicity of the theobromine derivatives.The utilized models are Ames mutagenicity (A-P), FDA carcinogenic effect in female rats (FDA-C-FR), carcinogenic doses (as the median) TD 50 in mice (M-MC-TD 50 ), maximum tolerated doses in rats by feeding (MTD-R-F), oral lethal dose LD 50 in rats (LD 50 -R), rats chronic lowest-observed-adverse-effect levels (CLOAEL-R), and potential ocular (OI) and topical (TI) irritations.
The results (Table 5), and most of the theobromine derivatives showed low toxicity profiles.Interestingly, 7 g exhibited safer patterns compared to sorafenib in three models (M-MC-TD 50 , LD 50 -R and CLOAEL-R) and a similar safe pattern in four models (A-P, FDA-C-FR, TI, and OI).However, in the MTD-R-F model, 7 g showed safe but with a less value than sorafenib.

Conclusion
In conclusion, the study has successfully designed and evaluated seven novel semisynthetic derivatives of theobromine with potential inhibitory activity against VEGFR-2.In particular, compound 7 g showed the most potent anti-VEGFR-2 activity (IC 50 ¼ 0.072 mM) and demonstrated dose-dependent inhibitory activity against MCF-7 (IC 50 ¼ 19.35 mM) and HepG2 (IC 50 ¼ 27.89 mM) cancer cells, with high selectivity indices.SAR study revealed that the substitution at the 3position of the hydrophobic tail with a methyl group is more advantageous than that with a methoxy group.Moreover, the substitution at the 4-position with electron-donating groups is more preferred than the substitution with electron-withdrawing groups.Further mechanistic studies indicated that compound 7 g induced G2/M phase cell cycle arrest, promoted apoptosis, and modulated immunomodulatory effects in MCF-7 cells.Molecular docking analysis and molecular dynamics simulations confirmed the stability of the VEGFR-2_7g complex, indicating the potential suitability of these compound as a drug candidate.Overall, these findings suggest that theobromine derivatives, particularly compound 7 g, could be anticancer lead compounds with VEGFR-2 inhibitory activities.Further biological as well as chemical modification studies for these compounds are demanded.The chemical modifications will comprise the change of substitution pattern in the terminal hydrophobic tail.In addition, the orientation of the linker moiety will be changed from the para-disusbtituted pattern into the metadisubstituted one.

In vitro antiproliferative activity
The antiproliferative activities of the synthesized compounds against HepG2 and MCF-7 cell lines were carried out using the MTT procedure as shown in Supplementary data (Al-Rashood et al., 2020;Denizot & Lang, 1986;Elkady et al., 2022).

In vitro VEGFR-2 inhibition
The VEGFR-2inhibitory activities of the synthesized compounds were tested using a VEGFR-2 ELISA kit according to the reported method described in Supplementary data (El-Adl et al., 2021;Elkady et al., 2022).

Figure 2 .
Figure 2. Pharmacophoric features of sorafenib as a prototype of the VEGFR-2 inhibitors.

Figure 3 .
Figure 3.The rationale of the presented design.

Figure 4 .
Figure 4. Cell cycle analysis of A) MCF-7 and B) 7 g/MCF-7 at the concentration of 19.35 mM.

Figure 9 .
Figure 9. A) RMSD values from the Trajectory for the VEGFR-2 protein (blue line), 7 g (green line), and VEGFR-2 & 7 g complex (red line), B) radius of gyration, C) SASA, D) change in the number of hydrogen bonds, E) RMSF, F) distance from the center of mass of 7 g and VEGFR-2 protein, G) shows a comparison between the ligand at 12.8 ns (cyan sticks) and 92.2 (green sticks).

Figure 10 .
Figure 10.Different energetic analyses of MM-GBSA and their values.Bars represent the standard deviations.

Figure 12 .
Figure 12.Active VEGFR-2's amino acids and the types of interactions with 7 g during the whole simulation time using the ProLIF python library.

Figure 13 .
Figure 13.The number and types of interactions detected using the PLIP webserver for each of the representative frames produced from clustering.Grey dashed lines: hydrophobic interactions, blue solid lines: hydrogen bonding, green dashed line: Pi-stacking interactions.

Table 1 .
In vitro cytotoxic activities of the target compounds against HepG2 and HCF-7 cell lines and in vitro inhibitory activities against VEGFR-2.

Table 2 .
Cell cycle analysis of 7 g/MCF-7 at the concentration of 19.35 mM.
$: means significant P value from MCF-7 group, significant P value < 0.05 & using unpaired Student's t-test.

Table 3 .
Gene expression analyses of 7 g/MCF-7 at the concentration of 19.35 mM.� : means significant P value from control group,.

Table 4 .
ADMET Screening values of the theobromine derivatives.