Development of Novel Class of Phenylpyrazolo[3,4-d]pyrimidine-Based Analogs with Potent Anticancer Activity and Multitarget Enzyme Inhibition Supported by Docking Studies

Phenylpyrazolo[3,4-d]pyrimidine is considered a milestone scaffold known to possess various biological activities such as antiparasitic, antifungal, antimicrobial, and antiproliferative activities. In addition, the urgent need for selective and potent novel anticancer agents represents a major route in the drug discovery process. Herein, new aryl analogs were synthesized and evaluated for their anticancer effects on a panel of cancer cell lines: MCF-7, HCT116, and HePG-2. Some of these compounds showed potent cytotoxicity, with variable degrees of potency and cell line selectivity in antiproliferative assays with low resistance. As the analogs carry the pyrazolopyrimidine scaffold, which looks structurally very similar to tyrosine and receptor kinase inhibitors, the potent compounds were evaluated for their inhibitory effects on three essential cancer targets: EGFRWT, EGFRT790M, VGFR2, and Top-II. The data obtained revealed that most of these compounds were potent, with variable degrees of target selectivity and dual EGFR/VGFR2 inhibitors at the IC50 value range, i.e., 0.3–24 µM. Among these, compound 5i was the most potent non-selective dual EGFR/VGFR2 inhibitor, with inhibitory concentrations of 0.3 and 7.60 µM, respectively. When 5i was tested in an MCF-7 model, it effectively inhibited tumor growth, strongly induced cancer cell apoptosis, inhibited cell migration, and suppressed cell cycle progression leading to DNA fragmentation. Molecular docking studies were performed to explore the binding mode and mechanism of such compounds on protein targets and mapped with reference ligands. The results of our studies indicate that the newly discovered phenylpyrazolo[3,4-d]pyrimidine-based multitarget inhibitors have significant potential for anticancer treatment.


Introduction
Cancer is a significant and prevalent health issue and is one of the leading causes of death globally [1,2].This disease is characterized by sustained proliferative potential, growth signals, and self-sufficiency, with apoptotic and antiproliferative cues resistance [3][4][5].There have been advancements in diagnosing cancer early and the development of new treatments; however, there is still a lack of effective therapeutics for treating advanced cancers [6].As conventional methods for treatment of cancer, radio and chemotherapy are no longer effective due to several side effects, including the unbiased destruction of body cells at comparable rates [7,8].Thus, multiple attempts have been made to treat advanced cancer cases utilizing targeted therapies [9,10].
The advancement of targeted therapies that aim to hinder or obstruct crucial cellular pathways in tumor growth and metastasis has led to a greater understanding of the diversity within tumors and their ability to circumvent the blockade of signaling pathways.Consequently, certain tumors may inherently display resistance or develop resistance to therapies that target a particular pathway.To address this challenge, employing a comprehensive strategy that involves the concise inhibition of multiple signaling pathways may be fruitful.This strategy can help counteract tumor resistance by blocking potential alternative paths for tumor escape [5,[21][22][23][24][25][26][27].
Vascular endothelial growth factor (VEGF) and the epidermal growth factor receptor (EGFR) are complementary pathways that play a fundamental role in tumor survival and diffusion [28][29][30].The VEGF signaling pathway is upregulated by the expression of EGFR, which contributes to cancer resistance.VEGF and EGFR can exaggerate tumors through the exertion of both indirect and direct effects on tumor cells [28].Targeting both pathways via mono-or multi-target therapeutics demonstrates a potential clinical benefit in many cancerous conditions.It is worth noting that therapeutics that impact VEGF-related pathways may contribute to the therapeutic targeting of the EGFR pathway [21,[31][32][33][34]. Dual inhibitors of VEGF/EGFR could potentially improve antitumor efficacy and overcome resistance.
1H-Pyrazolo [3,4-d]pyrimidine scaffold has been reported to be an essential pharmacophore in many anticancer agents [16,35,36], including EGFR-TKIs.Herein, given examples include compounds 1-8 (Figure 1), which were tested as anticancer agents with a pyrimidine-based library of the anti-EGFR-TK mechanism and have been approved by the FDA; these include first-generation examples such as erlotinib 1 and gefitinib 2; second-generation examples such as afatinib 3 and canertinib 4; third-generation EGFR-TKIs such as rociletinib 5, and avitinib 6; and clinical-phase compounds such as sapitinib 7 and dacomitinib 8.
The promising biological effects of 1H-pyrazolo [3,4-d]pyrimidine, in which the core scaffold was decorated with a pendant N-linker bonded to the aryl moiety at the 4 position, are able to consistently conserve activity against targets.
Other complexed examples of pyrazolo [3,4-d]pyrimidine-based EGFR-TK inhibitors (9-13) were discovered, with different potency profiles based on the ATP pharmacophore model.These compounds showed very interesting anticancer activity against specific cancer cell lines [37][38][39][40] (Figure 2).The promising biological effects of 1H-pyrazolo [3,4-d]pyrimidine, in which the core scaffold was decorated with a pendant N-linker bonded to the aryl moiety at the 4 position, are able to consistently conserve activity against targets.

Structure-Based Scaffold and Compound Design
Reports revealed that EGFR-TK is a polypeptide chain and that the ATP-binding groove of EGFR-TK is a 1186-amino-acids polypeptide chain.The ATP-binding region is composed of three areas: extracellular, intracellular, and hydrophobic regions (Figure 3a) [41,42].SAR analysis of EGFR-TKIs revealed that they have four common pharmaco-

Structure-Based Scaffold and Compound Design
Reports revealed that EGFR-TK is a polypeptide chain and that the ATP-binding groove of EGFR-TK is a 1186-amino-acids polypeptide chain.The ATP-binding region is composed of three areas: extracellular, intracellular, and hydrophobic regions (Figure 3a) [41,42].SAR analysis of EGFR-TKIs revealed that they have four common pharmacophoric features.First, the core structure consists of a flat nitrogenous heterocycle.This ring occupies the adenine binding pocket and forms hydrogen bonding with Met793, Thr790, and Thr854.The next feature is represented by a terminal hydrophobic head (a plain or substituted phenyl group) that interacts with hydrophobic region I.Most of the reported inhibitors also bear a middle secondary amine group, occupying the linker region between the adenine binding region and the hydrophobic region.The final feature, represented by a hydrophobic tail, connects to the flat hetero aromatic ring system which occupies hydrophobic region II.Based on the previous findings, we aimed to synthesize new 1H-pyrazolo [3,4-d]pyrimidine derivatives with potential anticancer activity.The target of this work was the synthesis of new derivatives carrying the same essential pharmacophoric features of the reported EGFR-TKIs as compound 1 and compound 7 (Figure 3b).We used a bioisosteric replacement strategy to fill up the binding pocket in EGFR-TKIs at four different positions (Figure 3b).In the first position, we used 1H-pyrazolo [3,4-d]pyrimidine as a flat heterocyclic system as a bioisostere of the quinazoline core on compounds 1 and 7.The suggested 1H-pyrazolo [3,4-d]pyrimidine core can fill the bulky space of the adenine-binding region Based on the previous findings, we aimed to synthesize new 1H-pyrazolo [3,4-d]pyrimidine derivatives with potential anticancer activity.The target of this work was the synthesis of new derivatives carrying the same essential pharmacophoric features of the reported EGFR-TKIs as compound 1 and compound 7 (Figure 3b).We used a bioisosteric replacement strategy to fill up the binding pocket in EGFR-TKIs at four different positions (Figure 3b).In the first position, we used 1H-pyrazolo [3,4-d]pyrimidine as a flat heterocyclic system as a bioisostere of the quinazoline core on compounds 1 and 7.The suggested 1H-pyrazolo [3,4d]pyrimidine core can fill the bulky space of the adenine-binding region (Figure 3a) [43,44].The nitrogen atoms at the core would engage several hydrogen bonds, which would be translated into excellent EGFR-TK potency [44,45].
The second position and third regions consisted of two terminal hydrophobic regions represented by phenyl group position 1 and alkyl or aryl side chains connected to a nitrogenous spacer.The last part was the nitrogenous linker (spacer) region with various lengths and heteroatom contents (amine, hydrazide, or thiosemicarbazide).The suggested modifications we envisioned led to study the SAR of this scaffold as an anticancer agent through the inhibition of EGFR-TK.

Anticancer Assay
The antiproliferative activity of the synthesized library was examined against three human cancer cell lines; breast (MCF-7), colon (HCT-116), and liver (HepG2) utilizing an MTT assay with the human diploid fibroblasts (WI-38) normal cell line as a comparison [46][47][48].The data are summarized in Table 1.According to our design, the tested compounds are classified into four categories based on the length and the chemotype of the spacer (Figure 3).In the first category, we used monosubstituted hydrazone linker.Among the tested derivatives in this category, compound 5b with p-hydroxyphenyl showed the most promising activity against the three cell lines when compared with the reference drug.Additionally, the O-methylated congeners, compounds 5d and e, maintained promising activity against HCT-116 cells with IC50 9.87 and 8.15 µM, respectively.The second category is represented by monosubstituted hydrazone linker.In this category, compound 5a, carrying the unsubstituted phenyl group, demonstrated noticeable activities against the three cell lines.Finally, interaction of the hydrazinyl derivative (3) with several isothiocyanates, namely, ethyl isothiocyanate, propyl isothiocyanate, butyl isocyanate, vinyl isothiocyanate, and phenyl isothiocyanate, in butanol (20 mL) under reflux afforded the N-alkyl/aryl-2-(6-methyl-1-phenyl-1H-pyrazolo [3,4-d]pyrimidin-4-yl)hydrazinecarbothioamides (9a-e), respectively, as shown in the Scheme 2.

Anticancer Assay
The antiproliferative activity of the synthesized library was examined against three human cancer cell lines; breast (MCF-7), colon (HCT-116), and liver (HepG 2 ) utilizing an MTT assay with the human diploid fibroblasts (WI-38) normal cell line as a comparison [46][47][48].The data are summarized in Table 1.According to our design, the tested compounds are classified into four categories based on the length and the chemotype of the spacer (Figure 3).In the first category, we used monosubstituted hydrazone linker.Among the tested derivatives in this category, compound 5b with p-hydroxyphenyl showed the most promising activity against the three cell lines when compared with the reference drug.Additionally, the O-methylated congeners, compounds 5d and e, maintained promising activity against HCT-116 cells with IC 50 9.87 and 8.15 µM, respectively.The second category is represented by monosubstituted hydrazone linker.In this category, compound 5a, carrying the unsubstituted phenyl group, demonstrated noticeable activities against the three cell lines.Possessing various chemical characteristics on scaffold dramatically ameliorated cytotoxic activity.In the third category, we tethered the linker to the pyrimidine ring to provide the triazolo[4,3-c]pyrimidine core.Using this strategy, we aimed to reduce rotation at this part to reduce the entropic penalty of the binding of the compound to EGFR-TK.The three derivatives showed moderate cytotoxic activities.Next, we used four heterocyclic substitutions to build the fourth category of our compounds (6d-7).None of these molecules exhibited any meaningful activity against the three cell lines.In the last category, we used a thiosemicarbazide linker connected to various aliphatic and aromatic side chains (9a-e) with prominent activity for analogs 9a and 9e.Collectively, this series displayed the best activity against the three tested cell lines in comparison with the other categories (Table 1).In addition, compound 9a, with ethyl side chain, overrides the activity of Doxorubicin against the HCT-116 cell line.In addition, normotoxicity was assessed for the most active analogs-5b, 5i, and 9e-on the WI38 cell line, and they exhibited a very low effect, with IC50 greater than 35 µM, which indicated a good safety profile.
Next, we investigated the cytotoxic activity of selected derivatives against the same cell lines using the extended treatment strategy.Using this strategy, we aimed to examine the resistance ability of the tested cell lines to our compounds after incubation for a long time.We realized that in comparison with doxorubicin, some tested compounds exhibited a time-dependent cytotoxic activity; for example, compound 5i.On the other hand, we noted a transient partial loss of cytotoxic activity 48 h post-treatment, followed by improved activity in the last time point.Table 2 and Figure 4 summarize the relative cytotoxic activity of the selected derivatives.Possessing various chemical characteristics on scaffold dramatically ameliorated cytotoxic activity.In the third category, we tethered the linker to the pyrimidine ring to provide the triazolo[4,3-c]pyrimidine core.Using this strategy, we aimed to reduce rotation at this part to reduce the entropic penalty of the binding of the compound to EGFR-TK.The three derivatives showed moderate cytotoxic activities.Next, we used four heterocyclic substitutions to build the fourth category of our compounds (6d-7).None of these molecules exhibited any meaningful activity against the three cell lines.In the last category, we used a thiosemicarbazide linker connected to various aliphatic and aromatic side chains (9a-e) with prominent activity for analogs 9a and 9e.Collectively, this series displayed the best activity against the three tested cell lines in comparison with the other categories (Table 1).In addition, compound 9a, with ethyl side chain, overrides the activity of Doxorubicin against the HCT-116 cell line.In addition, normotoxicity was assessed for the most active analogs-5b, 5i, and 9e-on the WI38 cell line, and they exhibited a very low effect, with IC50 greater than 35 µM, which indicated a good safety profile.
Next, we investigated the cytotoxic activity of selected derivatives against the same cell lines using the extended treatment strategy.Using this strategy, we aimed to examine the resistance ability of the tested cell lines to our compounds after incubation for a long time.We realized that in comparison with doxorubicin, some tested compounds exhibited a time-dependent cytotoxic activity; for example, compound 5i.On the other hand, we noted a transient partial loss of cytotoxic activity 48 h post-treatment, followed by improved activity in the last time point.Table 2 and Figure 4 summarize the relative cytotoxic activity of the selected derivatives.Possessing various chemical characteristics on scaffold dramatically ameliorated cytotoxic activity.In the third category, we tethered the linker to the pyrimidine ring to provide the triazolo[4,3-c]pyrimidine core.Using this strategy, we aimed to reduce rotation at this part to reduce the entropic penalty of the binding of the compound to EGFR-TK.The three derivatives showed moderate cytotoxic activities.Next, we used four heterocyclic substitutions to build the fourth category of our compounds (6d-7).None of these molecules exhibited any meaningful activity against the three cell lines.In the last category, we used a thiosemicarbazide linker connected to various aliphatic and aromatic side chains (9a-e) with prominent activity for analogs 9a and 9e.Collectively, this series displayed the best activity against the three tested cell lines in comparison with the other categories (Table 1).In addition, compound 9a, with ethyl side chain, overrides the activity of Doxorubicin against the HCT-116 cell line.In addition, normotoxicity was assessed for the most active analogs-5b, 5i, and 9e-on the WI38 cell line, and they exhibited a very low effect, with IC 50 greater than 35 µM, which indicated a good safety profile.
Next, we investigated the cytotoxic activity of selected derivatives against the same cell lines using the extended treatment strategy.Using this strategy, we aimed to examine the resistance ability of the tested cell lines to our compounds after incubation for a long time.We realized that in comparison with doxorubicin, some tested compounds exhibited a time-dependent cytotoxic activity; for example, compound 5i.On the other hand, we noted a transient partial loss of cytotoxic activity 48 h post-treatment, followed by improved activity in the last time point.Table 2 and Figure 4 summarize the relative cytotoxic activity of the selected derivatives.

In Vitro Cancer Related Targets Inhibition Analysis
Multi-targeting is a useful anticancer strategy with superior therapeutic attributes.A variety of tumors, such as aggressive breast cancers, offer the overexpression of different cellular enzyme targets which are responsible for large tumor size, poor differentiation, and poor clinical outcomes [49][50][51][52].Triazole hybrids were discovered to be multi-target EGFR WT -, EGFR T790M -, VEGFR-2-, and Topo II based-inhibitors, and they were evaluated for anticancer activity [53,54].Moreover, examples of Pyrazolo [3,4-d]pyrimidine-based multitarget anticancer inhibitors were reported [55].As seen in the data for the cytotoxicity of the newly synthesized analogs, only active candidate compounds 5b, 5i, and 9e were tested against a panel of cancer-related targets, namely, EGFR, VEGFR-2, and Topo-II, compared to reference drugs.The results are summarized in (Table 3).We found that compound 5b strongly and selectively inhibited EGFR activity in the lower range, with 20fold target selectivity indices over VGFR-2 and 108-fold over Topo-II.Moreover, compound 5i showed low selective and potent inhibition of EGFR WT and EGFR T790M over VGFR-2 and Topo-II, with 20/66 selectivity indices.Compound 9e was moderately active against VGFR-2 and Topo-II targets, with selectivity indices of 21/31 over EGFR WT .Overall, the scaffold pyrazolo [3,4-d]pyrimidine analogs showed different degrees of multitarget potent inhibitory activity, which might contribute to the discovery of anticancer agents.

Cell Cycle Analysis
Based on its antiproliferative activity, compound 5i was designated for further studies to explore its effect on the induction of apoptosis in the A549 cell line [56].We used this assay to elucidate the relationship between the proliferation inhibition and the cell cycle arrest, s well as to determine the biological phase in which the molecule interferes with cell growth.The cells were treated with 1 µM of 5i, and we used DMSO as a negative control.As presented in Figure 5, the data showed obvious interference with the native cell cycle distribution.Exposure of MCF-7 cells to our compound caused a decrease in the proportion of cells in the G0/G1 phase (from 57.39% to 49.63% when compared with the control).Moreover, it showed a slight increase in cell percentage in the S phase (from 33.97% to 43.12%), accompanied by a slight increase in the percentage of cells at the G2/M phase of the cell cycle (from 8.64% in the control to 7.25%) and a significant increase in the pre-G1 phase of the cell cycle (from 1.79% in the control to 36.06%).Collectively, these results indicated that compound 5i can lead to apoptosis through arresting the G1/S phase of cell cycle.

Annexin V-FITC Apoptosis Assay
We used annexin V-binding studies via flow cytometer to confirm the apoptosis in duction by our compound.The apoptotic nature of 5i against MCF-7 cells was tested vi flow cytometry detection after double-staining with Annexin V-FITC and propidium iodid (PI) [57].The results demonstrated that the treatment of A549 cells with our compound fo 48 h increased the early apoptosis ratio (lower right quadrant of the cytogram) from 0.44% to 12.62% and increased the late apoptosis ratio (higher right quadrant of the cytogram from 0.17% to 19.11%, indicating that 5i can induce MCF-7 cells apoptosis.In addition, treat ment of MCF-7 cells with our compound for 24 h resulted in 36.06% of apoptotic cells (earl + late) versus 1.79% of apoptotic cells in the untreated control (Figure 6).These result demonstrated that 5i might inhibit cell growth through cell apoptosis induction.

Annexin V-FITC Apoptosis Assay
We used annexin V-binding studies via flow cytometer to confirm the apoptosis induction by our compound.The apoptotic nature of 5i against MCF-7 cells was tested via flow cytometry detection after double-staining with Annexin V-FITC and propidium iodide (PI) [57].The results demonstrated that the treatment of A549 cells with our compound for 48 h increased the early apoptosis ratio (lower right quadrant of the cytogram) from 0.44% to 12.62% and increased the late apoptosis ratio (higher right quadrant of the cytogram) from 0.17% to 19.11%, indicating that 5i can induce MCF-7 cells apoptosis.In addition, treatment of MCF-7 cells with our compound for 24 h resulted in 36.06% of apoptotic cells (early + late) versus 1.79% of apoptotic cells in the untreated control (Figure 6).These results demonstrated that 5i might inhibit cell growth through cell apoptosis induction.

EGFR/VGFR2 Target Docking Simulations
The integration of experimental and computational methods is an attractive strategy by which to design and optimize successful drug candidates [32,34,51,52].Based on the biological activity of 5i, we carried out molecular docking studies as a crucial step to understand the mode of interaction of our selected molecule.First, a docking study was performed against EGFR (PDB ID: 1M17) (Figure 7a).Our compound was compared with erlotinib (compound 1, Figure 1), the known EGFR inhibitor.Previous studies [34,58] have indicated that erlotinib causes EGFR inhibition by binding to the site occupied by ATP during phosphotransfer.The N 1 of the quinazoline accepts the hydrogen bond from the Met769 amide nitrogen.It has been reported that 1-diphenyl-4,5-dihydro-1H-pyrazolo [3,4-d]pyrimidin-6-amine scaffold has successfully tolerated the EGFR pocket [59].Our data indicated that compound 5i conserved the same interaction with Met769 as the reference molecule.The N 1 of the pyrazole ring formed a hydrogen bond with Met 769, with a distance of 0.8 Å.Furthermore, the pyrazolo [3,4-d]pyrimidine moiety was incorporated into pi-pi interaction with Gly772 and Cys773, respectively.The peripheral phenyl group of 5i was buried inside hydrophobic region I. Additionally, a computational docking study was performed against VEGFR-2 (PDB ID: 3EWH) in comparison with a pyridyl-pyrimidine benzimidazole inhibitor (Figure 7b).The binding pattern of our molecule includes the pi-pi interaction of the core phenyl group with Leu1035 and the pyrimidine moiety with Lys868.Moreover, the hydrazide linker showed hydrogen bonding with Glu885, with a distance of 2.86 Å.This interaction imitates the same binding of the reference molecule with Glu885.A comparison of our molecule with both reference molecules through 2D molecular alignment revealed that 5i occupies the same space in both receptors.In addition, a mapping experiment on an active 5i analog referencing selective EGFR and VGFR-2 compounds led to the discovery of conserved molecular motifs that contributed strongly to the binding mechanism and hence to biological activity (Figure 8).

EGFR/VGFR2 Target Docking Simulations
The integration of experimental and computational methods is an attractive strategy by which to design and optimize successful drug candidates [32,34,51,52].Based on the biological activity of 5i, we carried out molecular docking studies as a crucial step to understand the mode of interaction of our selected molecule.First, a docking study was performed against EGFR (PDB ID: 1M17) (Figure 7a).Our compound was compared with erlotinib (compound 1, Figure 1), the known EGFR inhibitor.Previous studies [34,58] have indicated that erlotinib causes EGFR inhibition by binding to the site occupied by ATP during phosphotransfer.The N 1 of the quinazoline accepts the hydrogen bond from the Met769 amide nitrogen.It has been reported that 1-diphenyl-4,5-dihydro-1H-pyrazolo [3,4d]pyrimidin-6-amine scaffold has successfully tolerated the EGFR pocket [59].Our data indicated that compound 5i conserved the same interaction with Met769 as the reference molecule.The N 1 of the pyrazole ring formed a hydrogen bond with Met 769, with a distance of 0.8 Å.Furthermore, the pyrazolo [3,4-d]pyrimidine moiety was incorporated into pi-pi interaction with Gly772 and Cys773, respectively.The peripheral phenyl group of 5i was buried inside hydrophobic region I. Additionally, a computational docking study was performed against VEGFR-2 (PDB ID: 3EWH) in comparison with a pyridyl-pyrimidine benzimidazole inhibitor (Figure 7b).The binding pattern of our molecule includes the pi-pi interaction of the core phenyl group with Leu1035 and the pyrimidine moiety with Lys868.Moreover, the hydrazide linker showed hydrogen bonding with Glu885, with a distance of 2.86 Å.This interaction imitates the same binding of the reference molecule with Glu885.A comparison of our molecule with both reference molecules through 2D molecular alignment revealed that 5i occupies the same space in both receptors.In addition, a mapping experiment on an active 5i analog referencing selective EGFR and VGFR-2 compounds led to the discovery of conserved molecular motifs that contributed strongly to the binding mechanism and hence to biological activity (Figure 8).

Prediction of Drug-Likeness and ADME Properties
We used the OSIRIS Property Explorer to predict any potential side effects of our molecules, such as mutagenic, tumorigenic, irritant, and reproductive effects.Moreover, we tested a group of drug-relevant properties, including cLogP, LogS (solubility), MW, druglikeness, and overall drug-score.The in silico physicochemical and toxicological analyses were carried out with the OSIRIS Property Explorer program with respect to the two potent active analogs, 5b and 5i, compared to the two reported anticancer drugs.Properties with a high risk of undesirable effects, such as mutagenicity, tumorigenicity, irritant effects, and effects on reproductive physiology, as well as drug-relevant properties, including cLogP, LogS (solubility), MW, drug-likeness, and overall drug-score, are scored and color-coded, as shown in Table 4.The data of toxicity risks (shown in colors as red (high risk) and green (zero risk)) indicate a behavior consistent with the compound.Interestingly, the potential drug-likeness values of compounds 5i and 5b (4.26 and 3.97) were significantly higher than the two reference drugs, which showed negative values of −4.2 and −6.73.However, the in silico prediction of the OSIRIS Property Explorer showed that the introduction of 4-OH on the aryl ring can retain the lack of tumorigenic and mutagenic toxicity risk and enhance the druggability of compound 5i compared to the low-active one.In addition, we can see that both 5i and 5b compounds present a very excellent safety profile against all side effects.Generally, the drug-score values of compounds 5b and 5i (0.66 and 0.58) were better than those of sorafenib and erlotinib (0.20 and 0.38).It was noted via %ABS that analog 5i was 85% better than the others.The reasons for this good analysis are the absence of reactive functional groups within the chemical structure, the chemical stability, and the simplicity in the formula.

Prediction of Drug-Likeness and ADME Properties
We used the OSIRIS Property Explorer to predict any potential side effects of our molecules, such as mutagenic, tumorigenic, irritant, and reproductive effects.Moreover, we tested a group of drug-relevant properties, including cLogP, LogS (solubility), MW,

Conclusions
In this work, we applied a well-established chemical approach to the design and synthesis of phenylpyrazolopyrimidine-based analogs.In the protocol, we synthesized four different series of hydrazide, methylhydrazide, closed ring systems, and thiourea derivatives of high accessibility and good yields.The resulting compounds were evaluated for cytotoxic activity using an MTT assay against three malignant cell lines.Moreover, exemplary compounds were selected for mechanistic analysis using EGFR, VGFR-2, and Top-II enzymatic assay and cell cycle and apoptotic analyses.Most of the synthesized derivatives exhibited greater potency and selectivity performance than the reference inhibitor.Compounds 5b, 5i, and 9e were the most potent analogs, with IC 50 values of 3-10 µM cytotoxicity.Moreover, a detailed mechanistic analysis on the cancer cell line revealed that when compared to the positive control medication, most of the compounds had stronger antiproliferative activity.In MCF-7 cancer cells, compound 5i also enhanced apoptosis, produced cell cycle arrest at the G2/M phase, and caused DNA fragmentation.Molecule 5i appears to offer a lot of potential as a novel multi pyrazolopyrimidine-based lead compound for the identification of new anticancer medicines that target EGFR/VGFR-2 enzymes, based on our findings.In future studies, we intend to keep optimizing this molecule to create chemical entities with great anticancer activity and better selectivity to be advanced to preclinical studies.We will also use this molecule as a starting point from which to develop a combination therapy.All solvents and reagents used in this work were utilized as received from suppliers unless otherwise noted.Melting points were determined on a Stuart TM digital melting point apparatus (Stone, UK) and are uncorrected.The IR spectra were recorded on a Jasco FT/IR 460 plus spectrophotometer using KBr discs.The 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded on a Varian Mercury VXR-400 NMR 300 MHz using DMSO-d 6 as a solvent, and mass spectra were measured using a Hewlett Packard 5988 spectrometer.Elemental analysis was conducted at the Regional Centre for Mycology and Biotechnology (RCMP), Al-Azhar University, Cairo, Egypt.Reaction progress and purity of the synthesized molecules were monitored via thin-layer chromatography (TLC), utilizing Merck precoated silica gel 60 F 254 aluminum sheets.The reported yields refer to isolated compounds after purification.All spectral data are present in Supplementary Materials.

Figure 1 .
Figure 1.Examples of different generations of EGFR-TK inhibitors.

Figure 3 .
Figure 3. Work design.(a) Structure of ATP binding pocket, (b) Mapping of reference EGFR-TK inhibitors to pharmacophore points.

Figure 3 .
Figure 3. Work design.(a) Structure of ATP binding pocket, (b) Mapping of reference EGFR-TK inhibitors to pharmacophore points.

Figure 4 .
Figure 4. Relative cytotoxic activity of the selected molecules upon incubation with the selected cell lines for 48 h (a), 72 h (b), and 24 h (c).

2 Figure 5 .
Figure 5. Dot plot of Annexin V/PI double staining of MCF-7 control and treatment with analogu 5i.Statistical analysis of the apoptosis percentage of MCF-7 cells after incubation with compound 5i in conc.(3.81 µM) for 24 h.The data are reported as the mean ± SD of three independent exper ments in triplicate.

Figure 5 .
Figure 5. Dot plot of Annexin V/PI double staining of MCF-7 control and treatment with analogue 5i.Statistical analysis of the apoptosis percentage of MCF-7 cells after incubation with compounds 5i in conc.(3.81 µM) for 24 h.The data are reported as the mean ± SD of three independent experiments in triplicate.

Figure 6 .
Figure 6.DNA content distribution histograms of control and treated cells.Statistical analysis of cell cycle phases percentage of MCF-7 cells after incubation with compound 5i (3.81 µM) for 24 h.The data are reported as the mean ± SD of three independent experiments in triplicate.

Figure 6 .
Figure 6.DNA content distribution histograms of control and treated cells.Statistical analysis of cell cycle phases percentage of MCF-7 cells after incubation with compound 5i (3.81 µM) for 24 h.The data are reported as the mean ± SD of three independent experiments in triplicate.

Figure 8 .
Figure 8.Molecular mapping of compound 5i (middle) to reference bound ligands within protein targets.Color coding of similar fragments: red, green, and cyan types.

Table 1 .
Results of in vitro anticancer activity of the newly synthesized pyrazolopyrimidine compounds, IC 50 (µM).

Table 1 .
Results of in vitro anticancer activity of the newly synthesized pyrazolopyrimidine compounds, IC50 (µM).

Table 1 .
Results of in vitro anticancer activity of the newly synthesized pyrazolopyrimidine compounds, IC50 (µM).

Table 1 .
Results of in vitro anticancer activity of the newly synthesized pyrazolopyrimidine compounds, IC50 (µM).

Table 1 .
Results of in vitro anticancer activity of the newly synthesized pyrazolopyrimidine compounds, IC50 (µM).

Table 2 .
In vitro cytotoxic activity of selected compounds 48 and 72 h post-incubation.

Table 2 .
In vitro cytotoxic activity of selected compounds 48 and 72 h post-incubation.

Table 2 .
In vitro cytotoxic activity of selected compounds 48 and 72 h post-incubation.Data are presented as average IC 50 ± SD (µM) values for at least three experiments.