1,2,4-Triazole-Tethered Indolinones as New Cancer-Fighting Small Molecules Targeting VEGFR-2: Synthesis, Biological Evaluations and Molecular Docking

Targeting the VEGFR-2 signaling pathway is an inveterate approach toward combating pancreatic and hepatocellular cancers. Based on Sunitinib, the FDA-approved VEGFR-2 inhibitor, novel indolin-2-one-triazole hybrids were designed and synthesized as anti-hepatocellular and anti-pancreatic cancer agents with VEGFR-2 inhibitory activity. All the targeted compounds were assessed for their anti-cancer activity, revealing IC50 values extending from 0.17 to 4.29 µM for PANC1 and 0.58 to 4.49 µM for HepG2 cell lines. An extensive SAR study was conducted to explore the effect of different substituents along with N-alkylation. The potent anti-cancer analogs 11d, 11e, 11g, 11k and 14c were evaluated for their VEGFR-2 inhibitory actions, where their IC50 values ranged from 16.3 to 119.6 nM compared to Sorafenib, which revealed an IC50 of 29.7 nM, having compound 11d as the most active analog. An in silico ADME study was performed to confirm the drug-likeness of the synthesized compounds. Finally, molecular docking simulation was conducted for the most potent VEGFR-2 inhibitor (11d), demonstrating the strong binding with the vital amino acid residues of the VEGFR-2 ATP binding site.


Introduction
Angiogenesis is the process of recruiting new capillary blood vessels from existing ones.The viability of mammalian cells relies on oxygen and nutrients, prompting them to position themselves at distances of 100 nm to not more than 200 mm from blood vessels, which signifies the oxygen diffusion limit.In order to support the growth of multicellular organisms beyond this threshold, angiogenesis plays a crucial role in recruiting new blood vessels [1].This intricate process is regulated by a subtle equilibrium between pro-and anti-angiogenic molecules and can be disrupted in numerous diseases, notably cancer [2].Angiogenesis is switched on when pro-angiogenic factors surmount anti-angiogenic ones.Designated as one of the crucial hallmarks of cancer growth, metastasis and survival, angiogenesis blocking is entrenched as one of the foremost approaches to treating different life-threatening cancer diseases [3,4].The vascular endothelial growth factor family (VEGF) is one of the most prominent angiogenic activators, is highly expressed in various cancerous cells and plays a pivotal role in neovascularization [5].The VEGF family is responsible for activating the VEGF receptors (VEGFR-1, VEGFR-2 and VEGFR-3), which, in turn, enhances neoplastic vascularization [5].VEGFR-1 and VEGFR-2 are closely associated receptor tyrosine kinases and play pivotal roles in physiological and pathological angiogenesis, notably in the context of tumor angiogenesis.Despite being a kinase-impaired receptor tyrosine kinase, VEGFR-1 is actively involved.On the other hand, VEGFR-2, characterized by high activity, emerges as the principal VEGF receptor present on the surface of vascular endothelial cells [3].
The VEGF/VEGFR-2 pathway has gained great attention as a potent therapeutic target in multiple cancers [6].Since the up-regulation of VEGFR-2 is pronounced in malignant tumors, VEGFR-2 inhibitors have proved their profound anti-proliferative activities in many tumors, including pancreatic [7,8] and hepatocellular cancers [9,10].Siegel et al. reported that pancreatic cancer is the third foremost cause of death from cancer in both genders [11].In addition, liver cancer has had the speediest increase in mortality for decades.The current survival rate is lowest for both pancreatic (12%) and liver (21%) cancers.Obviously, 90% of liver cancer cases are stated to be hepatocellular carcinoma (HCC) [12].Advanced HCC is resistant to both chemotherapy and radiotherapy [13].Similarly, pancreatic cancer also develops chemoresistance, which hinders the effectiveness of the treatment, imparting poor response and quick relapse [14].These findings urge the need to develop novel chemotherapeutic agents of greater potency and selectivity with no resistance from the targeted cancerous cells.
Myriad indolin-2-one-based small molecules were developed and evaluated for their anti-proliferative activities, along with VEGFR-2 inhibition [15].The choice of indolin-2one scaffold is based on its superior pharmacokinetic properties, ease of synthesis and previously reported anti-tumor activity.This is clearly evinced by having a close look at Sunitinib's chemical structure.Sunitinib is a marketed VEGFR inhibitor with excellent anti-tumor activities against gastrointestinal stromal tumors, neuroendocrine tumors, renal cell carcinoma and pancreatic and breast cancers, while it is primarily an indolin-2-one derivative [16].Ornatinib is another chemotherapeutic agent indicated for hepatocellular carcinoma in phase 3 clinical trials with significant VEGFR-2 inhibition, which features an indolin-2-one ring [17].Moreover, Nintedanib is an indolin-2-one analog that acts as a tyrosine kinase inhibitor suppressing angiogenesis and completed Phase 3 clinical trials combined with Docetaxel to be used in non-small-cell lung cancer [18].On the other hand, the 1,2,4-triazole ring was explored for its anti-VEGFR-2 inhibition, where compound (I) showed VEGFR-2 inhibition with an IC 50 of 0.06 µM [19].In the same context, compound (II) exhibited noticeable cytotoxic activity against hepatocellular cell lines HepG2 (IC 50 = 10.02 µM) and manifested marked suppression of VEGFR-2 with IC 50 of 0.074 µM [20].A novel series of 1,5-diaryl-1,2,4-triazole urea signified its powerful anti-tumor activity through dual inhibitory action against both carbonic anhydrase and VEGFR-2, where compound (III) showed compelling VEGFR-2 inhibition with IC 50 26.3nM [21] (Figure 1).
The previously reported investigations of the binding mode of Sunitinib in VEGFR-2 revealed that the indolin-2-one ring accommodated the ATP binding site through the establishment of essential hydrogen bonds with Glu917 and Cys919 in the hinge region.Additionally, both the pyrrole ring and the fluoro group performed hydrophobic interactions that enhanced binding affinity.Finally, the diethylaminoethyl represents a hydrophilic tail toward the solvent-accessible region [22].
The strategy of combining biologically active molecules through molecular hybridization is a powerful technique in drug discovery.Its applications extend to various diseases, including AIDS, malaria, leishmaniasis and, of course, cancer.Interestingly, the molecular hybridization of biologically captivating pharmacophores, indolin-2-one included, is an ingrained strategy toward developing novel, potent chemotherapeutic agents that can effectively inhibit VEGFR-2 [23].The philosophy behind the hybridization is the reported supremacy of the hybridized pharmacophoric entities over the corresponding single ones.The previously reported investigations of the binding mode of Sunitinib in VEGFR-2 revealed that the indolin-2-one ring accommodated the ATP binding site through the establishment of essential hydrogen bonds with Glu917 and Cys919 in the hinge region.Additionally, both the pyrrole ring and the fluoro group performed hydrophobic interactions that enhanced binding affinity.Finally, the diethylaminoethyl represents a hydrophilic tail toward the solvent-accessible region [22].
The strategy of combining biologically active molecules through molecular hybridization is a powerful technique in drug discovery.Its applications extend to various diseases, including AIDS, malaria, leishmaniasis and, of course, cancer.Interestingly, the molecular hybridization of biologically captivating pharmacophores, indolin-2-one included, is an ingrained strategy toward developing novel, potent chemotherapeutic agents that can effectively inhibit VEGFR-2 [23].The philosophy behind the hybridization is the reported supremacy of the hybridized pharmacophoric entities over the corresponding single ones.Predominantly, the amalgamated compounds possess superior activity, fewer side effects, better pharmacokinetic properties and can evade drug resistance [24].
Inspired by the aforementioned findings and grounded on the potency shown by both indolin-2-one and 1,2,4-triazole counterparts and in a trial to raise potent anti-tumor agents with dominant anti-angiogenic activity, two series of indolin-2-one-1,2,4-triazole hybrids (11a-l and 14a-d) were designed grounded on the structure-activity relationship (SAR) of Sunitinib.The indolin-2-one scaffold was maintained in the designed compounds due to its importance in adopting the adenine binding pocket and forming the essential interactions with Glu917 and Cys919.To maintain the hydrophobic interactions of the fluorine atom in Sunitinib, it was kept in our designed compounds 11a-l with further use of other substituents (Cl, Br, OCH3 and NO2) to have deeper insight into their impacts on activity.Additionally, the pyrrole ring was bioisostered with a 1,2,4-triazole ring, where the triazole ring corroborated its potency in many VEGFR-2 inhibitors, as mentioned above.The linker was extended between the indolin-2-one and 1,2,4-triazole Inspired by the aforementioned findings and grounded on the potency shown by both indolin-2-one and 1,2,4-triazole counterparts and in a trial to raise potent anti-tumor agents with dominant anti-angiogenic activity, two series of indolin-2-one-1,2,4-triazole hybrids (11a-l and 14a-d) were designed grounded on the structure-activity relationship (SAR) of Sunitinib.The indolin-2-one scaffold was maintained in the designed compounds due to its importance in adopting the adenine binding pocket and forming the essential interactions with Glu917 and Cys919.To maintain the hydrophobic interactions of the fluorine atom in Sunitinib, it was kept in our designed compounds 11a-l with further use of other substituents (Cl, Br, OCH 3 and NO 2 ) to have deeper insight into their impacts on activity.Additionally, the pyrrole ring was bioisostered with a 1,2,4-triazole ring, where the triazole ring corroborated its potency in many VEGFR-2 inhibitors, as mentioned above.The linker was extended between the indolin-2-one and 1,2,4-triazole rings to be a hydrazide linker.Also, the N-substituted triazole ring with a p-F/Cl phenyl moiety was adopted in a trial hoping to increase the hydrophobic interactions and, consequently, the affinity of the targeted compounds to the ATP binding site.Besides, acetamide was used as the solvent-accessible tail instead of the diethylamino of Sunitinib (Figure 2).Furthermore, the impact of N-alkylation of the indolin-2-one with methyl and benzyl groups on biological activity was considered for exploration in compounds 14a-d.Eventually, the two series of hybrids (11a-l and 14a-d) were synthesized and biologically assessed for their growth inhibitory activities versus PANC1 and HepG2 cells.In addition, their VEGFR-2 inhibitory action was evaluated and expressed in terms of IC 50 .The ADME properties of the targeted molecules were calculated and evaluated to confirm their pharmacokinetic appropriateness.The final step involved a molecular docking study using the potent VEGFR-2 inhibitor among the test derivatives to figure out the binding modes that led to the inhibitory action.
assessed for their growth inhibitory activities versus PANC1 and HepG2 cells.In addi tion, their VEGFR-2 inhibitory action was evaluated and expressed in terms of IC50.The ADME properties of the targeted molecules were calculated and evaluated to confirm their pharmacokinetic appropriateness.The final step involved a molecular docking study using the potent VEGFR-2 inhibitor among the test derivatives to figure out the binding modes that led to the inhibitory action.

Chemistry
The synthesis of the targeted 1,5-diaryl substituted triazole-indolin-2-one hybrids 11a-l and 14a-d is illustrated in Schemes 1-3.4-Aminohippuric acid 4 was prepared by acylating the amino group of glycine amino acid 2 using p-nitrobenzoyl chloride 1 in aqueous sodium hydroxide solution to yield 4-nitrohippuric acid 3 and, subsequently the nitro group was reduced to the required amino group using Pd/C.To obtain com pound 5, 4-aminohippuric acid 4 was heated with acetic anhydride, resulting in the ac ylation of two distinct functional groups.The amino group was converted to the corre sponding acetamido group, while carboxylic acid was simultaneously activated to form an unstable mixed anhydride, leading to the formation of the azalactone ring containing an active methylene group.The active methylene was then coupled through the Kus kov-like reaction with freshly prepared diazonium salt 7a-b derived from 4-fluoro o

Chemistry
The synthesis of the targeted 1,5-diaryl substituted triazole-indolin-2-one hybrids 11a-l and 14a-d is illustrated in Schemes 1-3.4-Aminohippuric acid 4 was prepared by acylating the amino group of glycine amino acid 2 using p-nitrobenzoyl chloride 1 in aqueous sodium hydroxide solution to yield 4-nitrohippuric acid 3 and, subsequently, the nitro group was reduced to the required amino group using Pd/C.To obtain compound 5, 4-aminohippuric acid 4 was heated with acetic anhydride, resulting in the acylation of two distinct functional groups.The amino group was converted to the corresponding acetamido group, while carboxylic acid was simultaneously activated to form an unstable mixed anhydride, leading to the formation of the azalactone ring containing an active methylene group.The active methylene was then coupled through the Kuskov-like reaction with freshly prepared diazonium salt 7a-b derived from 4-fluoro or 4-chloroanilines 6a-b using sodium acetate salt to provoke hydrazone linker-tethered compounds 8a-b.Subsequently, the azalactone ring was opened and underwent Sawdey rearrangement [25] via refluxing in ethanol with hydrazine hydrate, ultimately resulting in the formation of key intermediates, hydrazides 9a-b, Scheme 1.
Hydrazides 9a-b underwent condensation with the 3-carbonyl group of diverse phenyl-substituted and N-alkylated indolin-2-one derivatives 10a-f and 13a-b, respectively, under reflux conditions in absolute ethanol, facilitated by the addition of a minimal catalytic quantity of glacial acetic acid to furnish hybrids 11a-l and 14a-d, Schemes 2 and 3.
4-chloroanilines 6a-b using sodium acetate salt to provoke hydrazone linker-tethered compounds 8a-b.Subsequently, the azalactone ring was opened and underwent Sawdey rearrangement [25] via refluxing in ethanol with hydrazine hydrate, ultimately resulting in the formation of key intermediates, hydrazides 9a-b, Scheme 1. Hydrazides 9a-b underwent condensation with the 3-carbonyl group of diverse phenyl-substituted and N-alkylated indolin-2-one derivatives 10a-f and 13a-b, respectively, under reflux conditions in absolute ethanol, facilitated by the addition of a minimal catalytic quantity of glacial acetic acid to furnish hybrids 11a-l and 14a-d, Schemes 2 and 3. Hydrazides 9a-b underwent condensation with the 3-carbonyl group of diverse phenyl-substituted and N-alkylated indolin-2-one derivatives 10a-f and 13a-b, respectively, under reflux conditions in absolute ethanol, facilitated by the addition of a minimal catalytic quantity of glacial acetic acid to furnish hybrids 11a-l and 14a-d, Schemes 2 and 3.
Scheme 2. Synthesis of target 1,5-diaryl substituted triazole-indolin-2-one hybrids 11a-l.Reagents and conditions: (i) ethanol, acetic acid, reflux, 2 h.Scheme 2. Synthesis of target 1,5-diaryl substituted triazole-indolin-2-one hybrids 11a-l.Reagents and conditions: (i) ethanol, acetic acid, reflux, 2 h.The structural attributes of the newly synthesized hybrids 11a-l and 14a-d were observed to align precisely with the outcomes derived from both spectral and elemental analyses.The 1 H NMR spectra revealed the presence of distinctive NH protons.The signal associated with NH of the acetamido moiety appeared in the range of δ 10.20-10.26ppm, while the NH signal of the acylhydrazone linker was observed between δ 11.70 and 14.32 ppm.Additionally, in derivatives lacking an N-alkyl group, the NH signal specific to the indolin-2-one moiety was detected between δ 10.69 and 11.91 ppm.Fur-Scheme 3. Synthesis of target 1,5-diaryl substituted triazole-indolin-2-one hybrids 14a-d.Reagents and conditions: (i) acetonitrile, potassium carbonate, stirring, room temperature, 18 h.(ii) Ethanol, acetic acid, reflux, 2 h.
The structural attributes of the newly synthesized hybrids 11a-l and 14a-d were observed to align precisely with the outcomes derived from both spectral and elemental anal-yses.The 1 H NMR spectra revealed the presence of distinctive NH protons.The signal associated with NH of the acetamido moiety appeared in the range of δ 10.20-10.26ppm, while the NH signal of the acylhydrazone linker was observed between δ 11.70 and 14.32 ppm.Additionally, in derivatives lacking an N-alkyl group, the NH signal specific to the indolin-2-one moiety was detected between δ 10.69 and 11.91 ppm.Furthermore, a discernible aliphatic signal for the CH 3 group of the acetamido motif was observed within the range of δ 2.05-2.10ppm.
In the 13 C NMR spectra, the three carbonyl groups present in the newly synthesized hybrids were distinctly evident.The acylhydrazone C=O signal appeared between δ 154.73 and 155.89 ppm, while the C=O signal associated with the indolin-2-one scaffold was observed between δ 161.00 and 164.94 ppm.Additionally, the acetamido C=O signal appeared at the highest chemical shift range (δ 168.84-169.46ppm).The aliphatic methyl group of the acetamido group was identified in the range of δ 24.11-24.56ppm.Finally, highresolution electrospray ionization mass spectrometry (ESI-HRMS) spectra exhibited peaks corresponding to quasimolecular ions [M+H] + and [M+Na] + , confirming the successful synthesis of the target compounds.

Biological Evaluation 2.2.1. In Vitro Anti-Proliferative Activity against PANC1 and HepG2 Cell Lines
The anti-tumor activity of the targeted compounds was assessed against PANC1 and HepG2 cell lines.The examined human cancer HepG2 and PANC1 cell lines were obtained from the American Type Culture Collection (ATCC).The resulting IC 50 values revealed the remarkable inhibition of cell growth in both contexts, with values for PANC1 cell lines ranging from 0.17 to 4.29 µM and for HepG2 cell lines spanning from 0.58 to 4.49 µM.First, for series 11a-l, compounds 11c, 11f, 11g, 11h and 11l manifested exceptional antiproliferative activity against the PANC1 cell line with IC 50 values at the submicromolar level (0.98, 0.23, 0.77, 0.22 and 0.17 µM, respectively).Moderate activity was evidenced for compounds 11b, 11d, 11e, 11j and 11k with IC 50 values of 1.74, 1.16, 2.22, 1.68 and 1.78 µM, respectively, for the same cell line.The least anti-neoplastic activity against PANC1 was observed for compounds 11a and 11i, as their IC 50 were 4.29 and 3.75 µM, respectively.
On the other hand, potent anti-tumor activity was exhibited versus the HepG2 cell line by compounds 11d, 11e, 11g and 11k, displaying IC 50 values in the submicromolar range (0.73, 0.76, 0.71 and 0.73 µM, respectively).All other compounds in the series conveyed appreciable inhibition of HepG2 cell growth with IC 50 values ranging from 1.01 to 2.41 µM, with the exception of compound 11i, which showed an IC 50 of 4.49 µM.
To elucidate SAR, this series can be categorized into those containing N-(4-fluorophenyl) (11a-f) and N-(4-chlorophenyl) (11g-l).The most potent compound inhibiting PANC1 within the fluorinated phenyl group was 11f (IC 50 = 0.23 µM), while in the chlorinated phenyl group, it was 11l (IC 50 = 0.17 µM).Both compounds notably featured a 5-nitro group in the indolin-2-one ring.It is worth mentioning that 11l was the most potent inhibitor of cancerous cell growth in this study.For the HepG2 cell line, 11d (IC 50 = 0.73 µM) and 11g (IC 50 = 0.71 µM) emerged as the most effective in the fluorinated and chlorinated phenyl groups, respectively.Despite the disparity in the substitution pattern on the indolin-2-one ring between the optimal compounds in both subseries-where 11d features 5-bromo while 11g is unsubstituted-the second-ranked compounds in inhibitory efficacy against HepG2 from the two subseries are those incorporating 5-methoxy (11e and 11k).Their IC 50 values (0.76 µM and 0.73 µM, respectively) were very proximal to those of 11d and 11g.
For a more nuanced comparative analysis between the two subseries, a clear dominance was observed in the inhibitory activity of compounds containing chlorinated phenyl against both cell lines when the indolin-2-one was either unsubstituted (11g) or bears a methoxy (11k) or nitro (11l) group at position 5.When it featured a fluoro group (11h), the efficacy showed an 8-fold improvement against PANC1 compared to its counterpart with fluorinated phenyl (11b).However, a slight decrease is noted towards HeG2.On the contrary, the superiority of compounds containing fluorinated phenyl becomes conspicu-ously evident when position 5 in the indolin-2-one incorporates chloro (11c) and bromo (11d) groups.
Interestingly, N-methyl/benzyl derivatives (14a-d) unveiled outstanding anti-proliferative activity patterns against both PANC1 and HepG2 cell lines relative to the unsubstituted peers, with IC 50 values ranging from 0.2 to 4.05 µM for PANC1 and 0.58 to 1.63 µM for HepG2.The compelling analog 14c expressed distinctive potency against both PANC1 and HepG2 cell lines with IC 50 values of 0.2 and 0.58 µM, respectively, establishing it as the most effectual growth inhibitor against HepG2 in this study.Furthermore, compounds 14b and 14d divulged substantial growth inhibition of the PANC1 cell line with IC 50 values of 0.76 to 0.42, respectively.Concerning their activity versus HepG2 cell lines, both compounds displayed IC 50 values equal to 0.98 and 0.92 µM, respectively.However, compound 14a showed a slight increase in the IC 50 to be 4.05 for PANC1 and 1.63 for HepG2.
Judging from the displayed IC 50 values, the compounds featuring chlorinated phenyl (14c and 14d) demonstrate a clear superiority in inhibiting the tested cancer cell lines compared to those containing fluorinated phenyl (14a and 14b).This indicates the importance of having a larger group than fluorine with less of an electron-withdrawing effect to improve the anti-proliferative potency.By scrutinizing the IC 50 values presented in Table 1 to evaluate the impact of N-substitution on the indolin-2-one ring, the positive influence of this strategic approach on inhibiting the growth of both cancer cell lines is unmistakably evident.The IC 50 values against the two tested cell lines for series 14a-d were consistently lower than their counterparts from the first series (11a, 11d, 11g and 11j).The only exception was for HepG2 with compound 14b.Although its IC 50 value resided in the submicromolar level (0.98 µM), it marginally surpassed its counterpart 11d (IC 50 = 0.73 µM).When assessing the impact of N-substitution with a methyl or benzyl group, it becomes apparent that their effects on enhancing efficacy are closely comparable.For example, introducing N-methyl to compound 11g (IC50 = 0.77 µM) yielded compound 14c (IC50 = 0.2 µM), demonstrating nearly a 4-fold increase in inhibitory activity against PANC1.Similarly, incorporating N-benzyl into 11j (IC50 = 1.68 µM) generated 14d (IC50 = 0.42 µM), displaying the same magnitude of efficacy increase against PANC1.
In addition, we evaluated the lethal effects of the new indolin-2-one-triazoles 11e, 11d, 11g, 11k and 14c on the non-tumorigenic Vero cell line to determine their specificity  When assessing the impact of N-substitution with a methyl or benzyl group, it becomes apparent that their effects on enhancing efficacy are closely comparable.For example, introducing N-methyl to compound 11g (IC50 = 0.77 µM) yielded compound 14c (IC50 = 0.2 µM), demonstrating nearly a 4-fold increase in inhibitory activity against PANC1.Similarly, incorporating N-benzyl into 11j (IC50 = 1.68 µM) generated 14d (IC50 = 0.42 µM), displaying the same magnitude of efficacy increase against PANC1.
In addition, we evaluated the lethal effects of the new indolin-2-one-triazoles 11e, 11d, 11g, 11k and 14c on the non-tumorigenic Vero cell line to determine their specificity When assessing the impact of N-substitution with a methyl or benzyl group, it becomes apparent that their effects on enhancing efficacy are closely comparable.For example, introducing N-methyl to compound 11g (IC 50 = 0.77 µM) yielded compound 14c (IC 50 = 0.2 µM), demonstrating nearly a 4-fold increase in inhibitory activity against PANC1.Similarly, incorporating N-benzyl into 11j (IC 50 = 1.68 µM) generated 14d (IC 50 = 0.42 µM), displaying the same magnitude of efficacy increase against PANC1.
In addition, we evaluated the lethal effects of the new indolin-2-one-triazoles 11e, 11d, 11g, 11k and 14c on the non-tumorigenic Vero cell line to determine their specificity toward the tested cancer cells.Remarkably, the indolin-2-one-triazoles that were studied showed low toxicity toward the normal Vero cells, with IC 50 values of 8.35 ± 0.62, 11.74 ± 0.93, 10.65 ± 0.71, 13.22 ± 1.09 and 7.92 ± 0.48, respectively.This indicates that they have good selectivity indexes and a safe profile (Table 2).

VEGFR-2 Inhibitory Activities
For further exploration of the synthesized compounds' mechanism of action, a VEGFR-2 inhibition assay was conducted for compounds 11e, 11d, 11g, 11k and 14c using Sorafenib as the reference compound.Appealingly, compound 11d, bearing the electron-withdrawing group 5-Br, evinced greater VEGFR-2 inhibition over Sorafenib, where it expressed IC 50 of 16.3 nM, while Sorafenib's IC 50 was 29.7 nM.Moreover, compounds 11e, 11k and 14c demonstrated anti-VEGFR-2 activity at a level with Sorafenib where their IC 50 ranged from 41.3 to 53.8 nM.Noteworthy, the replacement of the electron-withdrawing group 5-Br in compound 11d with the electron-donating group 5-OCH 3 in compounds 11e and 11k resulted in a decrease in the anti-VEGFR-2 activity.Eventually, compound 11g showed fair VEGFR-2 inhibition with an IC 50 of 119.6 nM, Table 3. Impressively, compound 11d manifests superior activity over compound III, which is considered a structurally similar compound to ours (VEGFR-2 IC 50 = 26.3nM).Since compound III was previously reported to have better VEGFR-2 inhibitory activity over Sunitinib (IC 50 = 39.7 nM), this strongly imparts great confidence toward compound 11d to effectively halt VEGFR-2 activity.In conclusion, the tested compounds are considered potent VEGFR-2 inhibitors; however, other kinases can be targeted through these compounds.Kinase activity profiling can be attempted in our future work to confirm the compounds' selectivity versus VEGFR-2.Attempting to deduce SAR from the data available in Table 3, it can be stated that the presence of Br at position 5 in the indolin-2-one moiety (11d) enhanced the efficacy against VEGFR-2 by approximately 3-fold compared to the presence of OCH 3 at the same position (11e).Clear observations from the evaluated chlorinated analogs revealed that the presence of substitution at position 5 (11k) or on N1 (14c) of the indolin-2-one motif increased activity by approximately 3 and 2.25 times, respectively, compared to when the indolin-2-one ring was unsubstituted (11g).

In Silico ADME Study
In silico investigation of the pharmacokinetic properties together with the druglikeness of the synthesized compounds was performed using SwissADME [26].Fortunately, the achieved results demonstrate the drug-likeness of the active compounds where all the compounds were established to obey Veber rules with the exception of compounds 11f and 11l [27].However, a maximum of two violations of the Lipinski rule was observed with some compounds.Being an important concern towards drug-drug interactions, which results in undesirable adverse reactions, prediction of the inhibition of different cytochrome P isoforms was demonstrated in Table 4.Moreover, the ADME properties, including blood-brain barrier (BBB) permeation, human gastrointestinal absorption (HIA), as well as being P-glycoprotein substrate or not, are presented in the BOILED-Egg model (Figure 3).Compounds 11a-11d, 11g-11i, 14a and 14c revealed high GI absorption.Meanwhile, all the compounds are suggested to not be permeable to the BBB, indicating no CNS side effects.Also, they are not expected to be substrates for P-glycoprotein (with the exception of compound 11e).To evaluate the binding modes of the synthesized indolin-2-ones and establish correlations between their structural attributes and inhibitory efficacy, a molecular docking analysis was conducted on VEGFR-2, with the PDB ID 4ASD as the reference [28].The validation of the molecular docking protocol commenced with the re-docking of Sorafenib, the co-crystallized ligand, at the ATP active site.The minimal root mean square deviation (RMSD) values of 1.15 Å between the co-crystallized ligand and the docked pose signify a nearly identical superimposition, affirming the practicality of the employed configuration for the proposed docking experiment, Figure 4. Also, Figure 5 shows 11d located in the VEGFR-2 active site.Strikingly, derivative 11d was recognized as the most captivating VEGFR-2 inhibitor in this study.Hence, it was selected for docking into the VEGFR-2 active site.It is noteworthy to mention that compound 11d engaged in interactions characterized by the establishment of four hydrogen bonds.Specifically, three of these bonds originated from the triazole ring's N4, establishing connections with crucial residues (Lys868, Glu885 and Asp1046), while the fourth hydrogen bond formed between the terminal acetamide moiety's C=O and Arg1027.Additionally, the bromine atom exhibited participation in a halogen bonding interaction with Leu840.
Regarding hydrophobic interactions, an observable pattern emerged wherein all aromatic components of 11d, including the indolin-2-one motif and the two phenyl rings, interacted through π-alkyl interactions with multiple residues within the active site, namely Leu840, Val848, Ala866, Leu889, Val898, Val899, Val916, Cys919 and Leu1035, Figure 6.The strong interactions observed within the ATP binding site explain the superior activity observed for compound 11d over Sorafenib.Strikingly, derivative 11d was recognized as the most captivating VEGFR-2 inhibitor in this study.Hence, it was selected for docking into the VEGFR-2 active site.It is noteworthy to mention that compound 11d engaged in interactions characterized by the establishment of four hydrogen bonds.Specifically, three of these bonds originated from the triazole ring's N4, establishing connections with crucial residues (Lys868, Glu885 and Asp1046), while the fourth hydrogen bond formed between the terminal acetamide moiety's C=O and Arg1027.Additionally, the bromine atom exhibited participation in a halogen bonding interaction with Leu840.
Regarding hydrophobic interactions, an observable pattern emerged wherein all aromatic components of 11d, including the indolin-2-one motif and the two phenyl rings, interacted through π-alkyl interactions with multiple residues within the active site, namely Leu840, Val848, Ala866, Leu889, Val898, Val899, Val916, Cys919 and Leu1035, Figure 6.The strong interactions observed within the ATP binding site explain the superior activity observed for compound 11d over Sorafenib.Strikingly, derivative 11d was recognized as the most captivating VEGFR-2 inhibitor in this study.Hence, it was selected for docking into the VEGFR-2 active site.It is noteworthy to mention that compound 11d engaged in interactions characterized by the establishment of four hydrogen bonds.Specifically, three of these bonds originated from the triazole ring's N4, establishing connections with crucial residues (Lys868, Glu885 and Asp1046), while the fourth hydrogen bond formed between the terminal acetamide moiety's C=O and Arg1027.Additionally, the bromine atom exhibited participation in a halogen bonding interaction with Leu840.

Molecular Dynamics
The outcomes obtained from both biological and docking investigations established compound 11d as a promising anti-cancer agent due to its potent inhibition VEGFR-2.This prompted us to delve deeper into its properties through molecular dynamic simulations (MDSs) for more in silico insights.MDSs are preferred for their precision in assessing the stability of a protein-ligand complex compared to other computational methods.Taking advantage of this, we simulated the binding poses of 11d with VEGFR-2, retrieved from the docking stage, for 100 ns.To facilitate and validate comparison, the Apo form of the target and the crystal coordinates of VEGFR-2 bound to Sorafenib were included in the simulation.
Figure 7 illustrates the considerable dynamicity of the free VEGFR-2 protein, indicative of its role as a primary oncogenic protein.This was evident in the RMSD calculations, where unbound VEGFR-2 displayed RMSD values of 4.2 Å. Notably, compound 11d exhibited a remarkable capability to restrict the dynamic behavior of VEGFR-2, as evidenced by lower RMSD values of approximately 1.8 Å compared to Sorafenib's 1.6 Å.The RMSF values mirrored the RMSD findings, with Apo proteins' residues experiencing average fluctuations of 4.1 Å for VEGFR-2 (Figure 8).The binding of 11d and Sorafenib induced significant stability in VEGFR-2 residues, reducing their fluctuation to average On the other hand, Table 5 presents the docking score of compound 11d, along with other compounds (11e, 11g, 11k and 14c) that were tested for their VEGFR-2 inhibitory action.Compound 11d exhibited a score (S = −9.6 kcal/mol) that was comparable to Sorafenib (S = −9.5 kcal/mol) and higher scores than 11e, 11g, 11k and 14c, which had scores of −8.3, −7.1, −8.5 and 18.0 kcal/mol, respectively (Table 5).These results could justify 11d's superior inhibitory activity.

Molecular Dynamics
The outcomes obtained from both biological and docking investigations established compound 11d as a promising anti-cancer agent due to its potent inhibition of VEGFR-2.This prompted us to delve deeper into its properties through molecular dynamic simulations (MDSs) for more in silico insights.MDSs are preferred for their precision in assessing the stability of a protein-ligand complex compared to other computational methods.Taking advantage of this, we simulated the binding poses of 11d with VEGFR-2, retrieved from the docking stage, for 100 ns.To facilitate and validate comparison, the Apo form of the target and the crystal coordinates of VEGFR-2 bound to Sorafenib were included in the simulation.
Figure 7 illustrates the considerable dynamicity of the free VEGFR-2 protein, indicative of its role as a primary oncogenic protein.This was evident in the RMSD calculations, where unbound VEGFR-2 displayed RMSD values of 4.2 Å. Notably, compound 11d exhibited a remarkable capability to restrict the dynamic behavior of VEGFR-2, as evidenced by lower RMSD values of approximately 1.8 Å compared to Sorafenib's 1.6 Å.The RMSF values mirrored the RMSD findings, with Apo proteins' residues experiencing average fluctuations of 4.1 Å for VEGFR-2 (Figure 8).The binding of 11d and Sorafenib induced significant stability in VEGFR-2 residues, reducing their fluctuation to average RMSFs of 1.5 and 1.2 Å, respectively.In summary, both RMSD and RMSF calculations converge on the conclusion that compound 11d effectively inhibits VEGFR-2 by forming robust and stable interactions with the enzyme's active sites.
Pharmaceuticals 2024, 17, x FOR PEER REVIEW 13 of 22 RMSFs of 1.5 and 1.2 Å, respectively.In summary, both RMSD and RMSF calculations converge on the conclusion that compound 11d effectively inhibits VEGFR-2 by forming robust and stable interactions with the enzyme's active sites.

Chemistry
Two devices used for acquiring NMR spectra belong to Bruker (Billerica, MA, USA), namely the Avance III Smart Probe Spectrometers.One operates at 400 MHz (400 MHz 1 H and 101 MHz 13 C NMR) and the other at 700 MHz (700 MHz 1 H and 176 MHz 13 C NMR).Additionally, a JOEL instrument (JEOL, Tokyo, Japan), specifically the ECA-500 II spectrometer (500 MHz 1 H and 126 MHz 13 C NMR), was employed for some compounds.The chemical shift was recorded in parts per million (δ).In all samples, deuterated dimethylsulfoxide (DMSO-d6) was utilize13d as a solvent.The coupling constants (J) were measured in Hertz (Hz).The Bruker MicroTOF spectrometer was used to obtain HRMS.A Stuart melting point device was used to determine uncorrected melting points.All re-  RMSFs of 1.5 and 1.2 Å, respectively.In summary, both RMSD and RMSF calculations converge on the conclusion that compound 11d effectively inhibits VEGFR-2 by forming robust and stable interactions with the enzyme's active sites.

Chemistry
Two devices used for acquiring NMR spectra belong to Bruker (Billerica, MA, USA), namely the Avance III Smart Probe Spectrometers.One operates at 400 MHz (400 MHz 1 H and 101 MHz 13 C NMR) and the other at 700 MHz (700 MHz 1 H and 176 MHz 13 C NMR).Additionally, a JOEL instrument (JEOL, Tokyo, Japan), specifically the ECA-500 II spectrometer (500 MHz 1 H and 126 MHz 13 C NMR), was employed for some compounds.The chemical shift was recorded in parts per million (δ).In all samples, deuterated dimethylsulfoxide (DMSO-d6) was utilize13d as a solvent.The coupling constants (J) were measured in Hertz (Hz).The Bruker MicroTOF spectrometer was used to obtain HRMS.A Stuart melting point device was used to determine uncorrected melting points.All re-

Chemistry
Two devices used for acquiring NMR spectra belong to Bruker (Billerica, MA, USA), namely the Avance III Smart Probe Spectrometers.One operates at 400 MHz (400 MHz 1 H and 101 MHz 13 C NMR) and the other at 700 MHz (700 MHz 1 H and 176 MHz 13 C NMR).Additionally, a JOEL instrument (JEOL, Tokyo, Japan), specifically the ECA-500 II spectrometer (500 MHz 1 H and 126 MHz 13 C NMR), was employed for some compounds.The chemical shift was recorded in parts per million (δ).In all samples, deuterated dimethylsulfoxide (DMSO-d6) was utilized as a solvent.The coupling constants (J) were measured in Hertz (Hz).The Bruker MicroTOF spectrometer was used to obtain HRMS.A Stuart melting point device was used to determine uncorrected melting points.All reaction chemicals and solvents were consumed without further purification after being purchased from commercial vendors.

Synthesis of 4-Aminohippuric Acid (4)
A solution of 4-nitrohippuric acid 3 (2.87 g, 12.8 mmol) in anhydrous methanol (40 mL) was prepared.Under N 2 atmosphere, Pd/C (10 wt% on activated carbon, 270 mg) was added.Reduction happened when temperature and H 2 pressure were adjusted at 25 • C and 1 bar, respectively.Subsequently, the reaction mixture was filtered off using a pad of celite, followed by the removal of methanol utilizing rotary evaporation to give 4-aminohippuric acid 4 as a white solid [33].Yield 74%; melt.pt.197-198 • C (reported melt.pt.199 • C [34]).4-Aminohippuric acid 4 (0.92 g, 4.73 mmol) was added to acetic anhydride (5 mL), then the temperature was elevated to reach 75 • C. The suspension was left for 40 min until it transformed into a yellowish-orange solution (solution A), which indicates the production of 5. Thereafter, the solution was allowed to cool to ambient temperature.In an ice bath 0-5 • C, 2 mL of 5N HCl was added to an appropriate aniline derivative 6a-b (3.67 mmol), then the mixture was left stirring for 20 min before 1 mL of an aqueous solution of NaNO 2 (0.33 g, 4.73 mmol) was added gradually to form the diazonium salts of aniline derivatives 7a-b.Fifteen minutes later, anhydrous sodium acetate (0.54 g, 6.59 mmol) was introduced to the reaction vessel (solution B).Thereafter, solution A was incrementally introduced to solution B and left to stir for 2 h at a temperature maintained between 0-5 • C. Flake ice was added to the mixture and the resulting product filtered and underwent thorough washing with distilled water (4 × 5 mL), methanol (2 × 3 mL) and petroleum ether (3 × 3 mL), followed by drying at 80 • C to afford acetamido-tethered hydrazones 8a-b (yield 73-80.3%)[21].

Figure 2 .
Figure 2. Rational design of the targeted molecules based on Sunitinib interactions with VEGFR-2 receptor.

Figure 2 .
Figure 2. Rational design of the targeted molecules based on Sunitinib interactions with VEGFR-2 receptor.

Figure 3 .
Figure 3. BOILED-Egg plot of the synthesized compounds.

2. 4 .
Molecular Modelling 2.4.1.Molecular Docking To evaluate the binding modes of the synthesized indolin-2-ones and establish correlations between their structural attributes and inhibitory efficacy, a molecular docking

Figure 3 .
Figure 3. BOILED-Egg plot of the synthesized compounds.

Pharmaceuticals 2024 ,
17, x FOR PEER REVIEW 11 of 22configuration for the proposed docking experiment, Figure4.Also, Figure5shows 11d located in the VEGFR-2 active site.

Figure 5 .
Figure 5. Binding of 11d with VEGFR-2, showing the whole protein and 11d located in the active site.

Figure 5 .
Figure 5. Binding of 11d with VEGFR-2, showing the whole protein and 11d located in the active site.

Figure 5 .
Figure 5. Binding of 11d with VEGFR-2, showing the whole protein and 11d located in the active site.

Figure 6 .
Figure 6.Docking representation of 11d within the active site of VEGFR-2 (A) 2D schematic presentation and (B) active site display.

Figure 6 .
Figure 6.Docking representation of 11d within the active site of VEGFR-2 (A) 2D schematic presentation and (B) active site display.

Table 1 .
In vitro anti-proliferative activities (IC50) for the synthesized indolin-2-one derivatives 11al and 14a-d against PANC1 and HepG2 cell lines.a IC50 values are presented as means ± S.D. for 3 separate experiments.

Table 1 .
In vitro anti-proliferative activities (IC50) for the synthesized indolin-2-one derivatives 11al and 14a-d against PANC1 and HepG2 cell lines.a IC50 values are presented as means ± S.D. for 3 separate experiments.

Table 2 .
Cytotoxic activities against non-tumorigenic Vero cell line.

, 11e, 11g, 11k and 14c.
a IC 50 values are the mean ± SD of three separate experiments.