Discovery of novel anticancer candidates based on a combination strategy of synthesis, characterization, biological activity evaluation, consensus docking and molecular modeling

Through the Kabachnic-Fields three-component reaction of 3(2-amino-acetyl)-quinazolin-4(3H)-one, compound III, various aromatic aldehydes, triphenyl phosphite, and lithium perchlorate as Lewis acid catalyst, new α-amino phosphonates molecules, IVa-f have been produced in a high yield. FT-IR, 1H-NMR, elemental analysis, and mass spectral data were used to determine the structures of the newly synthesized chemicals. Examined phosphonates, Iva–f, have been tested for their in vitro anticancer effects on the five cell lines HePG-2, MCF-7, Hela, HCT-116, PC-3, and normal cell, WI-38. Newly synthesized compounds' antioxidant activities were also covered. The novel-created α-amino phosphonate compounds have been evaluated on six cell lines and exhibit good anti-proliferative properties. The IVc molecule is the most effective antioxidant and anticancer candidate. Utilizing DFT/B3LYP/6-311G (d, p) method, the electronic and geometric characteristics derived from the stable structure of the studied compounds were examined. Additionally, there are outcomes for HOMO–LUMO, molecule electrostatic potential, and quantum chemical parameters. The stability of the most active phosphonate molecule, IVc, is attributed to hyper-conjugative interactions and charge delocalization. This was investigated using NBO analysis. Theoretical FT-IR and 1H-NMR measurements were applied to demonstrate the relationship between theory and experiment. An excellent concurrence between experimental and theoretical data was discovered. A docking simulation study was applied to forecast the inhibitory mode of action of the most active substance inside the cavity of estrogen receptor-positive (ER +) MCF-7 breast cancer.


Introduction
Heterocyclic chemistry contains at least half of all organic chemistry studies globally and is likewise referred to as the largest of classical organic synthesis [1,2]. More than 90% of new drugs include heterocyclic, which performs a crucial function in processes and industries as an interface between chemistry and biology [3]. Among all heterocyclic compounds, quinazolines are N-containing fused heterocyclic compounds with great utility in biological applications [4]. The quinazolin-4(3H)-one and its derivatives represent a vital class of fused heterocycles that possess a broad spectrum of biological activity, such as anticancer [5,6], antitumor [7][8][9][10][11], antioxidant [12], antimicrobial [13,14], antifungal [15], anticonvulsant [16,17], anti-HIV [18], anti-HCV [19] and anti-TMV [20] activities. The pharmacological properties and clinical applications of α-amino phosphonic acids and their corresponding α-amino phosphonates have been widely studied [21][22][23]. Their potential biological effects as potent enzyme inhibitors, antibacterial, anticancer, antioxidant, and antiviral drugs drew much attention [24][25][26][27][28][29]. The aminophosphonate group in the pharmacy core has been shown to increase anticancer action. Furthermore, many amino phosphonates have shown substantial inhibitory properties against human malignancies [30,31]. Quinazolinone derivatives and amino phosphonates have distinct biological and pharmaceutical properties. Therefore, structural changes of quinazolinone derivatives by inserting the α-aminophosphonate group boost pharmacological potency. [32][33][34][35]. In recent decades, advances in computational chemistry have partnered with experience in studying biological and biochemical structures and reactions to create a complete partner [36]. It has contributed significantly to understanding structure, molecular characteristics, processes, and reaction selectivity [36]. It also lets us comprehend the compound's likely behavior during reactions and provides crucial information about the compounds under investigation. These include total energy, binding energy, electronic energy, dipole moment, bond lengths, HOMO, and LUMO, and when this knowledge agrees with experimental evidence, its high value grows [37]. The applicability of semi-empirical methods/PM6 for calculating newly synthesized chemicals has been assessed [38,39]. Also, DFT theory is an important method used in calculating the properties of molecules [40][41][42][43]. The present work aims to synthesize new quinazoline compounds bearing phosphonate group by studying anticancer in vitro and antioxidant activity. In addition, the influence of changes in molecular and electronic structures on the biological activity of newly synthesized molecules was investigated using semi-empirical and DFT approaches. Also, see if we can find a link between the experimental and theoretical results.

Experimental
The compound melting points in degree centigrade through the capillary technique of Gallenkamp without correction have been suggested. The FT-IR spectra (KBr) on a Mattson 5000 FT-IR spectrophotometer, using a significant laboratory at TantaUniv, had been reported. Elemental analyses (C, H, and N) were accomplished at the Microanalytical Center of CairoUniv., Egypt. The 1 H-NMR spectra have been achieved at Kafr El-sheikhUniv., Egypt, on a BucherAC400 spectrometer working at four hundred MHz. The spectra have been recorded in DMSO-d6, and chemical shifts δ have been suggested relative to TMS. The MS had been recorded on Shimadzu Qp-2010 plus at the Microanalytical Center, CairoUniv. All samples were tested using analytical (TLC), which changed into accomplished on EM silica-gel F254 sheet, 0.2 mm. The biological evaluation was brought to the pharmacognosis department, Mans. Univ., Egypt.
Finally, excess pyridine is distilled out beneath decreased pressure; then, the solution is poured directly into a beaker containing beaten ice. It was filtered below suction, rinsed with ice-cold water in parts, and dried at 60 °C. The product was refined using ethanol recrystallization to produce a white crystalline solid of 3-(2-amino-acetyl)-quinazoline-4 (3H)-one, III, in an excellent yield (Fig. 1).
The compound structure III has been confirmed by: • Correct analytical data for C 10

Antitumor activity
The cytotoxic activity of the compounds under investigation against HepG2, MCF-7, Hela, HCT-116, PC-3, and WI-38 was briefly described utilizing doxorubicin as a reference anticancer medicinal medication (supplementary data).

Antioxidant assay
The bleaching of ABTS-produced radical cations and ascorbic acid as a standard antioxidant (positive control) were used to determine the antioxidant interest of the synthesized compounds (supplementary data).

Theory of calculations
The Gaussian 09W program packages created by Frisch and coworkers were used to execute all quantum chemistry calculations on a home computer [45]. The structure of the molecules is optimized using the semi-empirical/PM6 approach, then DFT with Beck's three-parameter exchange functional and nonlocal correlation functional, Lee-Yang-Parr, B3LYP [46][47][48] utilizing the 6-311G(d,p)basis set. Gaussian view 05 software) [49] was used to visualize the charge density distribution of the HOMO (Highest occupied molecular orbital), LUMO (Lowest unoccupied molecular orbital), and MEP (Molecular electrostatic potential). The consensus docking was carried out employing Molegro Virtual Docker (MVD) program [50].
Corrected elemental analysis (Experimental portion), FT-IR, 1H NMR, and mass spectroscopy were used to confirm the structures of compound III and α-aminophosphonate compounds IVa-f.
• Band within 2968 cm -1 corresponds to CH 2 aliphatic group. • The absorption band localized within 3043 cm -1 is due to CH aromatic stretching. • The stretching of C = C Aromatic and C-C Aromatic groups appears at 1613 and 1468 cm -1 , respectively. • Bands inside 1705 cm -1 is because of exocyclic C = O aliphatic , while a band at 1667 cm -1 is because of a C = O quinazoline ring. • A band at 1563 cm -1 is due to the C = N group. • Finally, the NH2 group is observed at 3155-3211 cm -1 .
• The CH 2 aliphatic protons produce a singlet signal at 2.52 ppm. • The NH 2 group is responsible for the broad singlet at 9.16 ppm, while the aromatic protons are resonated as multiplet in the 7.22-8.77 ppm region (Fig.S2).
• CH 2 aliphatic protons are responsible for a singlet signal at 2.50-2.2.53 ppm. • The CH-P proton causes a doublet signal around 4.95-5.95 ppm. • Multiplet signals at 5.96-8.3 ppm are attributed to multiplet aromatic protons, and the NH group is responsible for the singlet signal around 7.82-8.84 ppm (Table S2, Fig. S3).
The studied substances were characterized by elemental microanalysis, which revealed that the calculated value was extremely close to the estimated value. A molecular ion peak at m/z = 230 was confirmed in the mass spectrum of compound III (C 10 H 9 N 3 O 2 ), and a molecular ion peak at m/z = 570 was confirmed in the mass spectrum of compound IVa (C 29 H 23 N 4 O 7 P). The fragmentation sample is proven in Scheme S1. Also, a molecular ion peak at m/z = 541 was validated in the mass spectrum of compound IVb (C 29 H 24 N 3 O 6 P), and a molecular ion peak at m/z = 557 is corresponding compound IVc (C 29 H 24 N 3 O 7 P) (Scheme S2, Fig.S4).

Antitumor activity
The anticancer impact of 3(2-amino-acetyl)-quinazolin-4(3H)-one, III, and diphenyl [(aryl) ((2-oxo-2-(4-oxo-quinazolin-3(4H)-yl) ethyl)amino) methyl] phosphonate, IVa-f, in vitro determined using the standard MTT method [51,52] toward six consultant cell lines HePG-2, MCF-7, PC-3, HCT-116, Helaand Human lung fibroblast cell line, has been explained. Doxorubicin, a potent anticancer agent, is now a positive control. According to the obtained outcomes ( Fig. 4), each synthesized α-aminophosphonate has good, mild, or vulnerable anti-proliferative efficacy against the six cell lines tested as follows: 1. HePG-2: The compound IVc and IVf had a very high cytotoxicity activity, while the other compounds had moderate to mild cytotoxicity activity. 2. HCT-116: compounds IVc, IVb and IVf showed a powerful inhibitory potency, while the rest of the compounds showed moderate to weak activity. 3. PC-3: compound IVc and IVf demonstrated extraordinary anticancer activity, while the rest of the compounds showed moderate to weak activity. 4. MCF-7: compound, IVc, showed an extreme inhibitory efficiency, while compounds IVb and IVb showed moderate activity. The rest of the compounds showed moderate to weak cytotoxicity activity. . Hela: compound, IVc, showed extreme cytotoxicity activity, while the rest of the compounds showed moderate to weak activity. 6. WI-38: compounds with a hydroxyl group, IVb, IVc and IVf, have low cytotoxicity activity on the normal cell, and the other compounds have moderate cytotoxicity activity on the normal cell.
From the above data, we could conclude that compound IVc which contains 2, 4-diOH and compound IVf, which contain 3-OMe and 4-OH substituent, have the most potent derivatives against the five cancer cell lines (Table 1).

Structure-activity relationship (SAR) of compounds, IVa-f
The experimental cytotoxicity of the examined compounds to their structures was used to postulate a structure-activity relationship of the produced α-aminophosphonate derivatives: 1. The type of substituents in the aryl aldehyde moiety of α-amino phosphonates is essential for the wide range of cytotoxic activity against different cell lines (HePG-2, MCF-7, PC-3, HCT-116, Hela and WI-38). 2. Compound IVc is the most potent toward the five cell lines and showed a medium cytotoxic activity against the normal lung cell WI-38, which can be attributed to two electron-donating (OH) groups compared with doxorubicin which has OH moieties. 3. Because the number of OH moiety within the α-aminophosphonates ring system is reduced, compounds IVb and IVf are substantially less effective than compound IVc. 4. Presence of electron-donating OH moiety in the α-aminophosphonate compounds IVb and IVf improves potency more than the other aminophosphonate compounds (Table 1).

Antioxidant activity using DPPH inhibition and erythrocyte hemolysis of α-amino phosphonates, IVa-f.
Using the DPPH radical scavenging method, the total radical scavenging capability of the studied compounds was measured and compared to ascorbic acid [53,54] (Scheme 3). The data showed that the compound, IVc, has the most potent antioxidant activity and sturdy inhibitory activity within the hemolysis assay of all compounds (Tables S3  and S4). All synthesized compounds have potent inhibitory activity within the hemolysis assay, except compound IVb, which exhibits a weak inhibitory activity within the hemolysis assay (Table S4). Thus, the presence of substituents within the phenyl-substituted α-amino phosphonates appears to have influenced the antioxidant screening of the investigated compounds. When combined with a DPPH solution, the novel di-substituted heterocyclic IVc-amino phosphonates can donate a hydrogen atom. As a result, the release of protons from NH groups of DPPH radicals is facilitated [55]. As a result, this molecule can protect cells and tissues against oxidative stress caused by unstable radicals, leading to various disorders, including cancer.

Quantum chemical calculations
Quantum chemistry approaches and molecular modeling procedures can define various molecular components, including reactivity, shape, binding residences, molecular fragments, and substituents. The relationship between structural factors and the organic activity of α-amino phosphonates was investigated using quantum chemical calculations. The optimized molecular systems with the lowest energies obtained from the computations of the researched compounds are demonstrated in Fig. 5. Experimentally, it was discovered that compound IVc, which contains a di-substituent electron-donating OH groups within the aryl aldehyde moiety of the -amino phosphonates, had higher biological activity than the first quinazolinone compound, III, and the other phosphonates. Hence, quantum chemical calculations could be used for this rationalization. The compound IVc, which contains two OH groups, has the maximum HOMO energy (-5.996 eV), indicating that it should react as a nucleophile (electron donor) and increase its donation capability to the target protein, resulting in increased activity (Table 2).
∆E is a characteristic reactivity descriptor; decreasing the value of ∆E increases the reactivity of the compounds. The calculations revealed that compound IVc has a lower separation energy (3.484 eV) than compound III (4.602 eV), as well as the alternative phosphonates, and so has the best softness values, which could explain the compound's reactivity (Table 2). On the other hand, ∆E is a crucial quantity for estimating the molecular system's nonlinear optical homes (NLO). A low ∆E cost indicated an easy digital transition and better NLO homes with an appreciation of a famous molecule, particularly urea [56]. The examined ∆E values for all molecules are lower than urea's (6.884 eV), indicating that each of the produced phosphonates is a viable structure for photovoltaic devices such as solar cells. The IVe I Vf most range of electronic loads (ΔNMAX) acquired from the environment (donor) through the inhibitor (acceptor) is the measured reactivity index through energy stabilization. The calculation showed that compound IVc has a higher ΔNmax, 2.442e, than that of compound III, 2.060e (Table 2), which shows that it permits the charge transfer and alternate of electron density among the compound and protein and hence growth of the biological activity that is in an excellent settlement with the experimental data. The calculations confirmed that di-substituted phosphonate, IVc, has a higher dipole moment value, 4.821D, than compound III, 2.067D, and mono-substituted phosphonate, IVb, 3.465D (Table 2). This method shows that this compound is more extraordinarily polarizable (hydrophilic) and hence more tremendously reactive than III and IVb; this is pretty much in line with the results of the experiments. It was concluded from the above discussion that the computation calculations showed that the α-aminophosphonate compounds increase the reactivity more than the starting quinazolinone compound, which agrees well with the experimental data. Also, the computed values can predict that di-substituted electron-donating OH groups within the aryl aldehyde moiety of α-amino phosphonates are significantly more effective than one OH group. These might be extra favorable for the reactivity in the direction of the enzyme and consents properly with the experimental data ( Table 2).

The frontier molecular orbitals
Frontier molecular orbitals are incredibly beneficial as quantum chemical reactivity descriptors. The energy of the highest occupied molecular orbital (E HOMO ) is associated with an electron-donating capability. In comparison, the energy of the lowest unoccupied molecular orbital (E LUMO ) is strongly related to the prosperity of the molecule toward charge acceptance [57,58]. HOMO and LUMO charge density distributions for α-aminophosphonate molecules are shown in Fig. 6. It changed into proven that, for electronwithdrawing substituted phosphonates, IVb, HOMO is in particular localized on the 2 phenyl groups connected to the phosphorous atom. The LUMO is mainly delocalized on the nitrophenyl moiety (Fig. 6). However, the HOMO for compound IVc is located on one of the phenyl groups attached to the phosphorous atom and at the dihydroxy-substituted phenyl moiety. Thus, it can interact with the biological target as a nucleophile (hydrogen bond donor). The LUMO of IVc is selectively delocalized at the quinazolinone ring, which can be interacted with the biological target as an electrophile (hydrogen bond acceptor). In the case of mono-electrondonating substituted phosphonates, IVb, the HOMO's charge density distribution is concentrated on one of the phenyl groups attached to the phosphorus atom, as well as on the hydroxy-substituted phenyl moiety (act as a nucleophile with the target enzyme) and LUMO is specifically delocalized at the quinazolinone ring (act as electrophile with the target enzyme) (Fig. 6).

Molecular electrostatic potential
The molecular electrostatic potential (MEP) is strongly influenced by electronic density. MEP is one of the most essential and valuable computational tools for revealing the most reactive regions for electrophilic and nucleophilic attacks [59,60]. Different colors signify different values of the electrostatic potential on the surface. Red, orange, yellow, green, and blue indicate potential increases [61]. Red color regions of MEP represent the electrophilic reactive sites, while the nucleophilic reactive ones are represented by blue color (Fig. 6).
The negative ability covers the carbonyl moiety of the quinazolinone ring, the carbonyl moiety connected to the quinazoline ring, and the oxygen atoms connected to a phosphorous group. In comparison, the positive areas are above the remaining groups. The electrostatic interplay forces among the researched chemicals and the organic target can be defined from the MEP plots (Fig. 6).
All calculated values referenced to the TMS chemical shift at the same level of theory Chemical shifts, δ (ppm) 3-(2-amino-acetyl)-quinazolin-4(3H)-one, III  (2) O26 to σ*(C22-C23) and π*(C23-C24) transitions, which might be chargeable for the α-aminophosphonate molecules' intermolecular charge transfer (ICT), 5.92 and 29.47 kJ/mol, respectively, which were validated as stabilization energies. Also, the interaction of LP (1)O40 prolonged to σ*(N15-H48) denotes a bigger delocalization and stabilization of the entire system. The different critical intramolecular conjugative interaction is tabulated in Table 3. From the NBO evaluation results, it can be inferred that within the α-amino phosphonates, the presence of di-substituted electron-donating OH groups will boost the molecule's reactivity.

NMR spectral analysis
Nuclear Magnetic Resonance (NMR) spectroscopy has grown to be a vital research tool supplying accurate predictions of molecular geometries in present-day chemistry [65][66][67]. Computational NMR applications in diverse chemistry fields have been developed [68]. The computation of a few key NMR parameters, such as nuclear spin-spin couplings, shielding constants, and chemical shifts, is an active and essential part of theoretical research [69,70].  The gauge-independent atomic orbital (GIAO) is a massive upgrade for the calculation of the NMR parameters Hartree-Fock (HF) method [71]. In this study, 1 H-NMR chemical shift values of the quinazolinone, III and amino phosphonates, IVa-f, were calculated using HF/GIAO/6-31G method in DMSO solvent. Different NMR descriptors are listed in Table 5 [72]. Experimentally and theoretically generated 1 H-NMR isotropic shift values had been as compared, and a linear correlation with a high correlation coefficient was observed (Fig. 9). A good correlation between experimental 1 H-NMR chemical shift values and the GIAO-NMR approach was found.

Molecular docking results analysis for the most potent ligand, IVc
Molecular docking in the pharmaceutical enterprise is effective in silicon strategy for discovering novel therapies for unmet scientific desires predicting drug-target interactions. It no longer solely gives binding affinity between drugs and aims at the atomic level; however, it additionally elucidates the imperative pharmacological houses for specific medicines.
A molecular docking learn about used to be carried out to recognize the underlying mechanism of the examined molecule IVc toward MCF-7 breast most cancers mobile line. Molegro Virtual Docker (MVD) program performs binding ligand docking, so the most advantageous geometry of the ligand will be decided for the duration of the docking process. The 3D shape of the protein used to be acquired from the protein data bank using its unique (PDB code: 6p05) with a resolution of 1.54 Å [73]. A grid (x = 12.02, y = 13.71, z = 5.92 and r = 10 Å) is generated round the binding website already occupied through the co-crystallized ligand so that co-crystallized ligand can be excluded, and new compounds  can be connected to the identities binding site of the target protein.
The optimized molecular shape of the IVc molecule received from DFT/6-311G (d,p) was used as enter file for the docking calculations. The lowest energy docked conformer of IVc binds to the active website online residues with average binding energy, − 141.657 kcal/mol making intimate contacts with the active residence of the target enzyme thru significant bonded and nonbonded interactions. Suppose we disclose the binding mode of compound Ivc. It exhibited an appropriate docked score, − 23.021 kcal/ mol, and formation of six hydrogen bonds with catalytic amino acids, i.e. Asp88, Tyr97, Lys91, Gln85 and Trp81with oxygen (O18, O26, O46) and nitrogen (N7 and N15) atoms (Fig. 12). Figure 13 confirms 2D interplay plot of ER + -IVc complicated visualized the usage of LIGPLOT + [73].
The hydrophobic phenyl moiety formed two pi-interactions with hydrophobic residues, Leu92 and Leu94, with distances 2.63 and 3.74 Å (Fig. 14). These interactions better the stabilization of ER + -inhibitor complicated and provide an explanation for the highest inhibition of IVc, which consents nicely with the experimental biological activity. Also, a strong electrostatic interplay is shaped between the electronegative oxygen atom (O26) and necessary positively charged residue, Lys91, with a distance of 2.25 Å. This might also be one of the structural prosperity for a higher activity of IVc (Fig. 15). Figures 16 and 17 exhibit the surface illustration of the minimal energy shape and the secondary shape view of the docked pose of IVc in the binding web page of ER + .

Conclusion and outlook
Novel α-amino phosphonates-based quinazolinone rings were synthesized, and their structures were elucidated using different spectroscopic methods. The in vitro anticancer activities of diphenyl (aryl) [2-oxo-2-(4H-quinazolin-4-one-3-yl) ethyl-amino] methyl phosphonate, IVa-c, against six representative cell lines were explained. As a result, the di-substituted phosphonate compound, IVc, is the most active compound among all studied compounds. NBO analysis for IVc, the compound was calculated and confirmed that the presence of di-substituted Fig.12 The visual interaction of IVc and hydrogen bond interactions are indicated by dotted lines, with numerals indicating the distance in angstroms. Sticks represent protein, and balls and sticks designate IVc electron-donating OH moieties will increase the reactivity of the compound.
The theoretical FT-IR data were calculated to discover the compounds' characteristic vibration frequencies, which correlate well with experimental data. Also, a good correlation was found between experimental 1 H-NMR chemical shift values and the GIAO-NMR approach. Molecular docking calculation was performed for the most active molecule IVc using MVD software. The tested compound, IVc, could be a potent anticancer agent because it has a good docking score. Nitrogen, oxygen atoms and phenyl moieties form a hydrogen bond and hydrophobic and electrostatic interactions with crucial residues within the binding pocket. This study should assist in enhancing or finding new drugs for future anticancer agents.   Fig. 15 The electrostatic interaction between the O26 atom of a hydroxyl group (electronrich atoms) and the positively charged residue (Lys91). The molecular surface represents the protein, and the ligand is colored by their partial charges, where red is a negative charge and blue is a positive charge