Synthesis and screening of novel 2,4-bis substituted quinazolines as tubulin polymerization promoters and antiproliferative agents

Abstract

Twelve 2,4-bis-substituted quinazoline-based compounds were synthesized and screened for antiproliferative and tubulin polymerization enhancing potential. In the series, compound A4V-3 substituted with an imidazole ring displayed IC₅₀ values of 4.25 μM, 2.65 μM, and 9.95 μM, and A4V-5 with a benzotriazole substitution displayed IC₅₀ values of 3.45 μM, 7.25 μM, and 8.14 μM against MCF-7, HCT-116 and SHSY-5Y cancer cells, respectively. In the mechanistic studies involving cell cycle analysis, apoptosis assay and JC-1 studies, compound A4V-3 was found to arrest the cells in the G₂/M phase of the cell cycle and induce mitochondria-mediated apoptosis. In addition, compound A4V-3 displayed significant tubulin polymerization-enhancing potential. 2,4-Bis-substituted quinazoline-based compounds showed appreciable drug-like characteristics and can be developed as potent anticancer agents.

Twelve 2,4-bis-substituted quinazoline-based compounds were synthesized and screened for antiproliferative and tubulin polymerization enhancing potential.

1. Introduction

Cancer is one of the most serious health problems worldwide responsible for millions of deaths every year. Around 1 958 310 new cases and 609 820 million deaths were reported due to cancer in 2023 alone.¹ Abnormal gene functioning and mutated gene expressions play a crucial role in the progression of cancer. Major hallmarks associated with cancer cells such as tissue invasion, metastasis, apoptosis, angiogenesis, etc., have been identified and explored as significant targets for developing novel anticancer drugs.² Tubulin (α and β tubulin heterodimers) is a part of the cytoskeleton and it plays a vital role in cell division, cell signalling, and development and maintenance of cells. A dynamic equilibrium is maintained between α and β tubulin heterodimers and microtubules. During the mitosis phase of cell division, microtubules help correct chromosomes' segregation via mitotic spindle formation and any alteration in it may lead to cell cycle arrest.³ In many types of cancers, tubulin overexpression is observed, which leads to faster cell division/growth and promotes tumour formation.⁴ Thus, targeting tubulin protein is one of the most common approaches to target different types of cancers and to develop potent anticancer compounds. Tubulin proteins can be targeted either by microtubule-destabilizing agents like colchicine or by microtubule-stabilizing agents like paclitaxel.^5,6

Microtubule-stabilizing agents, upon binding to the taxane site, enhance microtubule stabilization and halt the depolymerization process. This disruption can interfere with spindle dynamics during cell division, preventing affected cells from progressing past the mitotic checkpoints and cause arrest at the G₂/M phase.⁷ Although the exact mechanism of action of microtubule-stabilizing agents has yet to be fully understood, ligands binding to the taxoid site exert significant effects on interactions at three key regions within tubulin: H3, S3, and the M loop. These regions play vital roles in the interactions between protofilaments during microtubule assembly. Ligand binding can enhance interactions between the M loop in the β-subunit and the H1-S2 and H2-S3 loops of neighbouring protofilaments, thereby promoting microtubule stabilization.^8,9 Tubulin binding agents (TBAs) are potent mitotic poisons that are broadly classified as microtubule-stabilizing agents (MSAs) like taxanes and epothilones and microtubule-destabilizing agents (MDAs) such as vincristine, vinblastine, and colchicine.¹⁰ Various microtubule-stabilizing agents in clinical practice like paclitaxel, docetaxel, ixabepilone and cabazitaxel (Fig. 1) have been widely used in the treatment of a variety of solid tumors including Kaposi sarcoma, triple-negative breast cancer and ovarian cancer.^11,12 These clinical agents have several drawbacks like low water solubility, poor pharmacokinetic properties, multidrug resistance, and toxicity (myelosuppression and mucositis) issues.^13,14

Fig. 1. Designing of compounds using US FDA-approved microtubule-stabilising agents and quinazoline-based anticancer agents.

A large number of structurally different synthetic and natural compounds have been explored against different cancer targets and many of them are under clinical practice.¹⁵ However, most of these drugs suffer with one or other limitations including multidrug resistance. Hence, there is a huge demand for the development of novel small molecules that can effectively treat cancer. A number of research groups are working to develop novel anticancer molecules with improved potency and efficacy against resistant cancer cells. The quinazoline ring system is a pharmacologically important scaffold which can be seen as a core moiety in various biologically active small molecules. Slight changes in the substitution patterns to the quinazoline nucleus can lead to distinguishable differences in the biological activities.¹⁶ The quinazoline moiety is widely found in antibacterial,¹⁷ antifungal,¹⁸ antiviral,¹⁹ anti-inflammatory,²⁰ analgesic,²¹ and antitumor agents.²² The presence of this active pharmacophore in many natural and synthetic compounds is found to be crucial for biological activity. In our previous studies and by others, it has been found that differently substituted quinazoline derivatives displayed potent cytotoxic effects against various cancer cell lines with minimal toxic effects on normal cells, making them excellent antitumor agents.^23,24

Tubulin inhibitors exhibit critical pharmacophoric characteristics that facilitate efficient engagement with the tubulin binding site. This encompasses hydrophobic groups that enhance interactions, hydrogen bond donors and acceptors for critical amino acid binding, and aromatic rings that promote π–π stacking interactions. Moreover, flexible linkers facilitate the appropriate spatial arrangement of these functional groups, ensuring optimal binding and interference with tubulin polymerization, thus inhibiting cell division. We designed the compounds based on USFDA-approved microtubule inhibitors that possess essential pharmacophoric features for binding to the receptor and quinazoline-based scaffolds as potent anticancer agents as shown in Fig. 1.

In the current research work, we have synthesized a series of quinazoline derivatives with symmetrical substitution at the 2nd and 4th position of the quinazoline ring. These compounds were evaluated for their anticancer potential, and most of them were effective against different cancer cell lines. Compounds A4V-3 and A4V-5 showed significant anticancer activity against MCF-7, HCT-116, and SHSY-5Y cancer cells. In mechanistic studies involving cell cycle analysis, apoptosis assays, and JC-1 studies, compound A4V-3 was found to arrest the cells in the G₂/M phase of the cell cycle and induce mitochondria-mediated apoptosis. In addition, compound A4V-3 displayed significant tubulin polymerization-enhancing potential as compared to the positive control, paclitaxel.

2. Results and discussion

2.1. Chemistry

The quinazoline-based target compounds were synthesized as per Scheme 1 by utilising synthetic methodologies reported by us and others. Substituted benzaldehydes were used as substrates for the synthesis of anthranilamide derivatives. Substituted quinazoline-2,4(1H,3H)-dione derivatives were synthesized using urea and anthranilamide derivatives. 2,4-Dichloroquinazoline derivatives were synthesized using phosphorus oxychloride, and in the final step different types of secondary amines such as morpholine, imidazole, triazole etc. were substituted at the 2nd and 4th position of 2,4-dichloroquinazoline derivatives. The crude products were purified through column chromatography. All the synthesized compounds were characterized using ¹H NMR, ¹³C NMR, and HRMS.

Scheme 1. Synthesis of quinazoline-based microtubule-stabilizing agents. Reaction conditions: (i) I₂, NH₄OH (40%), rt, (ii) conc. HNO₃/H₂SO₄; (iii) Fe/NH₄Cl, EtOH refluxing; (iv) NaOH, EtOH refluxing; (v) acetic acid, urea, DMSO, 100 °C; (vi) POCl₃, toluene, reflux; (vii) K₂CO₃, secondary amines, DMF, 80 °C.

2.2. Biological studies

2.2.1. Antiproliferative activity against cancer cell lines (MCF-7, HCT-116 and SHSY-5Y)

All the synthesized compounds (A4V-1 to A4V-12) were evaluated for anticancer activity against three different cancer cell lines, MCF-7 (breast), HCT-116 (colon) and SHSY-5Y (neuroblastoma) using standard MTT assay previously used by our group²⁵ (Table 1). Four different concentrations of each compound were used in triplicate and results were analysed after 48 h of compound treatment. Colchicine was used as a positive control while cells without test sample or positive control was considered as control.²⁶ Cell viability was assessed and antiproliferative activity was calculated in terms of IC₅₀ values recorded in Table 1.

Table 1. In vitro antiproliferative activity (IC₅₀ in μM) of compounds A4V-1 to A4V-12 against MCF-7, HCT-116 and SHSY-5Y cancer cell lines. Data are presented as the mean ± SDs of three independent experiments.

Code	R		MCF-7 (μM)	HCT-116 (μM)	SHSY-5Y (μM)
A4V-1	3,4,5-OCH3		24.12 ± 0.23	21.32 ± 0.19	>25
A4V-2	3,4,5-OCH3		18.76 ± 0.21	>25	15.89 ± 0.12
A4V-3	3,4,5-OCH3		4.25 ± 0.12	2.65 ± 0.08	9.91 ± 0.18
A4V-4	3,4,5-OCH3		23.12 ± 0.31	13.81 ± 0.28	>25
A4V-5	3,4,5-OCH3		3.45 ± 0.18	7.25 ± 0.10	8.14 ± 0.20
A4V-6	H		15.31 ± 0.30	>25	11.04 ± 0.25
A4V-7	H		11.45 ± 0.34	9.43 ± 0.27	15.74 ± 0.29
A4V-8	H		19.02 ± 0.16	17.54 ± 0.23	15.32 ± 0.27
A4V-9	H		14.61 ± 0.11	>25	18.93 ± 0.18
A4V-10	H		21.75 ± 0.29	>25	>25
A4V-11	H		12.76 ± 0.16	9.19 ± 0.20	>25
A4V-12	H		>25	20.65 ± 0.29	23.82 ± 0.14
Colchicine			4.48 ± 0.14	1.65 ± 0.08	6.82 ± 0.17

All the synthesized compounds of this series have a symmetrical substitution of secondary amine present at the 2nd and 4th positions of the quinazoline ring. From the literature and our previous studies, it was concluded that some groups like benzotriazole, imidazole, triazole, morpholine, and trimethoxy substitution over the quinazoline ring play a crucial role in anticancer activity.^24,27 In this series, compounds A4V-1 to A4V-5 contained a trimethoxy-substituted aromatic ring of the quinazoline scaffold, while in A4V-6 to A4V-12, the ring was unsubstituted. A4V-1, with methyl piperazine as a substituent on the quinazoline ring, showed moderate activity against MCF-7 and HCT-116 cell lines and was found ineffective against SHSY-5Y cells at 25 μM. Similarly, the morpholine ring (A4V-2) displayed moderate activity against MCF-7 and SHSY-5Y cell lines while no activity against HCT-116 cells. Imidazole functionality on the quinazoline ring (A4V-3) was found most effective against all the three cancer cell lines with IC₅₀ values of 4.25 μM, 2.65 μM and 9.91 μM against MCF-7, HCT-116 and SHSY-5Y cell lines, respectively. A4V-3 displayed comparable activity as shown by the standard inhibitor colchicine. The 1,2,4-triazole derivative (A4V-4) showed moderate anticancer activity against MCF-7 and HCT-116 but was inactive against SHSY-5Y even at 25 μM. A benzotriazole moiety on the quinazoline ring (A4V-5) displayed significant anticancer activity against all the tested cell lines with an IC₅₀ value in the range of 3 μM to 8 μM. Removal of the trimethoxy substituents from the quinazoline ring (A4V-6) led to decreased anticancer activity and the compound was found to be moderately active against MCF-7 and SHSY-5Y cell lines only. Similarly, A4V-7 with an imidazole substituent was found to be less active as compared to A4V-3. In contrast, A4V-8 with triazole functionality showed improved anticancer activity as compared to the corresponding trimethoxy derivative (A4V-4). Compound A4V-9 with a morpholine ring showed comparable activity with A4V-2 and displayed moderate anticancer activity against MCF-7 and SHSY-5Y but was inactive against HCT-116 at 25 μM concentration. Interestingly, it was observed that an extended substituent like benzylpiperazine was not found compatible for anticancer activity and A4V-10 was found inactive against HCT-116 and SHSY-5Y at 25 μM. Similarly, compound A4V-11 with a phenylpiperazine moiety and A4V-12 with a methylpiperazine moiety did not show any significant improvement in anticancer activity. Thus, in this series compounds A4V-3 and A4V-5 were found to be potent compounds against all the three cancer cell lines and were chosen for further mechanistic studies. The two most potent compounds A4V-3 and A4V-5 were evaluated for their cytotoxicity against normal cell lines (HEK 293) and both compounds were found to be nontoxic even at the highest concentration (25 μM) (Fig. 2).

Fig. 2. SAR studies from the cytotoxicity data.

2.2.2. Cell cycle study

In general, most of the colchicine binding site inhibitors and colchicine strongly arrest the cells at the G₂/M phase of the cell cycle.²⁸ Cell cycle analysis was performed against SHSY-5Y cells for the compounds A4V-3 and A4V-5 at 5.0 μM to determine whether the antiproliferative activity resulted from cell cycle arrest. It was found that A4V-3 displayed significant growth arrest at the G₂/M phase of the cell cycle (Fig. 3). In comparison to the control, wherein 50.1% of cells were arrested at the G₁ phase and 28.4% of cells were arrested at the G₂/M phase, A4V-3 showed 79.0% cell arrest at the G₂/M phase of the cell cycle. Colchicine exhibited a cell arrest of 90.0% at the G₂/M phase. In contrast, A4V-5 showed cell arrest of 78.9% at the G₁/G₀ phase of the cell cycle (Table 2).

Fig. 3. Cell cycle assay with propidium iodide at 5.0 μM concentration of the test compounds treated for 48 h with colchicine as a positive control.

Table 2. Percentage distribution of cells in the cell cycle study.

S. no.	Phase of the cell cycle	Control (%)	Colchicine (%)	A4V-3 (%)	A4V-5 (%)
1	G1/G0	50.1	5.1	11.3	78.9
2	S	16.2	4.1	6.8	7.7
3	G2/M	28.4	90.0	79.0	8.4

2.2.3. Apoptosis study by flow cytometry

A vast number of studies have demonstrated that tubulin polymerization inhibitors can activate cell apoptosis.²⁹ Hence, the active compounds A4V-3 and A4V-5 were tested for their ability to initiate apoptosis in SHSY-5Y cells by staining with Annexin V–FITC and propidium iodide. It was observed that treatment of cells with the test compounds at 5.0 μM initiated cellular apoptosis and disintegrated cellular integrity. It has been found that A4V-3 and A4V-5 showed late apoptotic cell death of 50.3% and 51.8%, respectively, as compared to 3.0% apoptosis of the non-treated cells. Colchicine as a positive control showed 54.5% apoptosis in late apoptosis after 48 h of treatment (Fig. 4). This showed that both A4V-3 and A4V-5 exhibited most of their antiproliferative activity due to induction of cellular apoptosis.

Fig. 4. Quantitative analysis of apoptotic cells using Annexin V–FITC/PI double staining and flow cytometry calculations.

2.2.4. JC-1 study

Mitochondria play an essential role in the mechanism of apoptosis so change in the mitochondrial membrane potential is the determining factor for whether a cell is healthy or not.³⁰ A fluorescence probe, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine), was used to analyse the change in mitochondrial membrane potential (Δψ_m) as this dye aggregates inside the healthy mitochondria and produces red/orange fluorescence. It is evident that many antimitotic compounds induce apoptosis through the mitochondria.³¹ Thus SHSY-5Y cells were treated with the lead compounds (A4V-3 and A4V-5) at 5 μM concentration. A4V-3 displayed moderate change in the mitochondrial membrane potential. As evident from Fig. 5, depolarization was observed for A4V-3 and hence the cytotoxicity displayed by this compound might be mitochondrial mediated. Meanwhile, for A4V-5, no depolymerization was observed and hence the cytotoxicity of this compound might be mediated through some other pathway.

Fig. 5. Treatment of SHSY-5Y cancer cells with A4V-3 and A4V-5 and their JC-1 analysis showing variations in the mitochondrial membrane potential (Δψ_m).

2.2.5. Tubulin polymerization activity

Taxane binding site agents arrest the cells in the G₂/M phase of the cell cycle by binding to the straight conformation of assembled tubulin and prevent depolymerization.³² This inhibition of conversion of straight to curved prevents the depolymerization of assembled tubulin heterodimer that ultimately leads to irregular mitotic spindles and metaphasic arrest of mitotic cells.³³ In the cell cycle, apoptosis and JC-1 studies, it appears that these compounds may interfere in the mitotic phase and induce apoptosis of cancer cells. In the cell cycle analysis, A4V-3 arrested the cells in the G₂/M phase; hence this compound was screened for its polymerization inhibition potential. Paclitaxel was used as a positive control as it enhances tubulin polymerization. Microtubule polymerization assays were performed using a fluorescence-based tubulin polymerization assay kit (Cytoskeleton, Denver, CO, USA). The analysis was performed for 60 min and data were recorded at time intervals of 1 min. To our surprise, A4V-3 showed polymerization-enhancing activity when compared with the control and paclitaxel (positive control).³⁴ As evident from Fig. 6, compound A4V-3 showed tubulin polymerization activity at 5 μM concentration, while at lower concentration (1 μM) it showed no polymerization (parallel line with the control). When compared with paclitaxel at 1 μM, A4V-3 displayed similar activity at 5 μM concentration (Fig. 6). Thus, it was concluded that A4V-3 showed significant tubulin polymerization potential.

Fig. 6. Effect of compound A4V-3, colchicine and paclitaxel on tubulin polymerization: tubulin polymerization was monitored by the increase in fluorescence at 360 nm (excitation) and 440 nm (emission) for 1 h at 37 °C.

2.3. Computational studies

2.3.1. Molecular docking and MM/PBSA binding free energy calculation

Taxane binding site agents enhance tubulin polymerization, arrest the cells in the G₂/M phase of the cell cycle and induce apoptosis that leads to cell death.³⁵ From all the in vitro experiments it is evident that compound A4V-3 displayed significant anticancer potential and arrested the cancer cells in the G₂/M phase of the cell cycle. In the tubulin polymerization assay, it was found that A4V-3 behaved like paclitaxel, i.e., it enhanced the polymerization of tubulin protein and acted as a promoter of tubulin polymerization. Thus, docking studies of A4V-3 were performed on the taxol binding site (TBS) of the tubulin protein. The interactions between the ligands and the receptor site were scored using Glide (GLIDE 12.3 module of Schrödinger Suite). The docking protocol was validated by docking of taxol into the crystal structure of tubulin protein on the taxol binding site. A4V-3 displayed a docking score of −4.63 kcal mol⁻¹ in the taxol binding site of the tubulin protein (PDB: 1JFF)³⁶ (Fig. 7A and B).

Fig. 7. 3D and 2D docking poses of A4V-3 (A and B) along with taxol (C and D) in the taxane binding site. A4V-3 and taxol showed docking scores of −4.63 kcal mol⁻¹ and −5.11 kcal mol⁻¹, respectively.

In the case of taxol, docking into the same cavity showed two hydrogen bond interactions which provide stability to the compound inside the cavity with ARG278 and GLU27 (Fig. 7C and D). The binding free energy for taxol was found to be −37.25 kcal mol⁻¹. The binding free energy, viz −40.44 kcal mol⁻¹, of A4V-3 and all these interactions and surrounding amino acids showed that compound A4V-3 fit well inside the cavity and supported the tubulin polymerizing activity of the compound.

2.3.2. Molecular dynamics trajectory analysis

Based on the docking score and biological screening, potential protein–ligand docked complexes with the reference compound were analysed for complex stability by 100 ns molecular dynamics (MD) simulation using GROMACS 2021.3. RMSD was calculated for a lead molecule with protein for about 100 ns, presented in Fig. 8A. The RMSD plot for the A4V-3–protein complex was found to be in the range of 0.25–0.28 nm without much deviation, while the RMSD for the paclitaxel–protein complex was found to be in the range of 0.25–0.33 nm. Similarly, the compactness value for the complex was recorded and found to be stabilized at ∼2.15 nm and ∼2.16 nm for A4V-3 and paclitaxel protein complexes, respectively (Fig. 8B). The RMSF value for protein was calculated when bound to the respective ligand and depicted a range of 0.1–0.5 nm (Fig. 8C). Higher fluctuations were observed in various regions such as 28–64, 171–181, 215–224 and 273–288 residues of protein, which might be due to the presence of loops in those regions. Meanwhile, the RMSF value for the ligand was found to vary between 0.025 and 0.21 nm and 0.025 and 0.35 nm for A4V-3 and paclitaxel, respectively (Fig. 8D).

Fig. 8. Molecular dynamics simulation trajectory (100 ns) analysis for the potential docked complex and reference via (A) RMSD and (B) rGy. (C) RMSF for protein. (D) RMSF for ligand.

Furthermore, the protein–ligand complex was assessed for hydrogen bond profiling throughout the simulation. The hydrogen bond occupancy for the ligands were observed to be 50–60% (Fig. 9A), wherein A4V-3 and paclitaxel showed 1–4 and 1–10 hydrogen bonds, respectively. Finally, the hydrogen bond distance distribution was also recorded and it was found that all the hydrogen bonds formed were within the applied cut-off value of 0.35 nm (Fig. 9B) for both complexes. Overall, the A4V-3 complex was found to be stabilized during the trajectory of 100 nm within the tubulin protein.

Fig. 9. Molecular dynamics simulation trajectory (100 ns) analysis for the potential docked complex and reference via (A) hydrogen bond formation and (B) hydrogen bond length distribution.

2.3.3. ADME studies

Schrödinger's QikProp module was used for ADME studies of the synthesized compounds. The QikProp module utilizes different parameters of Lipinski's rule of five for the analysis of drug-like properties. The lipophilicity of compounds is used to determine whether the molecule will cross the biological membrane, and for that log P (<5) is an important physiochemical property. For the standard taxol, it is 5.39, while A4V-3 displayed lipophilicity of 3.82. The lead compound displayed log P values of less than 5, which is within the limit for druggable compounds, indicating that the molecules are lipophilic in nature. The solubility of the drug candidates in biological systems is also an important parameter that must be considered and log S values for taxol and A4V-3 were found to be −6.86, and −3.78, respectively. It also indicates that the compounds displayed optimum aqueous solubility and there should be no issue of bioavailability during in vivo studies. The molecular weights of these compounds were less than 500 and the number of hydrogen bond donors and hydrogen bond acceptors also lies within the acceptable range (Table 3). Compound A4V-3 showed more than 95% human oral absorption as compared to taxol, which showed only 61.5%, and compound A4V-3 underwent a lesser number of metabolic reactions (metb). Thus, A4V-3 showed drug-like characteristics and may be considered for next-stage evaluation.

Table 3. Drug-like characteristics of A4V-3 as determined by QikProp application of Schrödinger.

Name	Mol. wt.	Binding energy/(kcal mol−1)	log P	log S	HB donor	HB acceptor	% human oral absorption	Dipole	#metb
A4V-3	352.13	−32.91	3.82	−3.78	1	5	96.25	5.68	3
Taxol	853.91	−61.90	5.39	−6.86	3	10	61.5	4.23	8

3. Conclusion

2- and 4-bis-substituted symmetrical secondary amine derivatives of quinazoline and trimethoxy-substituted quinazoline were synthesized. These compounds were evaluated for anticancer potential against MCF-7, HCT-116 and SHSY-5Y cancer cells. Most of the compounds showed mild to moderate anticancer activity but two compounds (A4V-3 and A4V-5) displayed significant anticancer activity against all the three cancer cell lines. These two potent derivatives were evaluated for cytotoxicity potential against normal cell line HEK-293 and both the compounds exhibited no toxicity even at 25 μM concentration. The two most potent derivatives (A4V-3 and A4V-5) were selected for further in vitro studies. In the cell cycle study, only A4V-3 arrested the cells in the G₂/M phase of the cell cycle and displayed significant apoptosis. Thus, A4V-3 was selected for the anti-tubulin study; this compound showed polymerization-enhancing activity and at 5 μM concentration its activity was found to be similar to that of paclitaxel at 1 μM. Thus, the interesting results obtained in this study may help in the identification of potent leads with microtubule-stabilizing activity and hence can be developed as anticancer agents. In in silico studies the lead molecule A4V-3 (−40.44 kcal mol⁻¹) was found to have higher binding free energy than the reference compound (−37.25) and also formed a stable complex with protein throughout the MD simulation of 100 ns.

4. Experimental

4.1. General

All the reagents were of AR/GR quality and purchased from Sigma-Aldrich, Loba-Chemie Pvt. Ltd., Sisco Research Laboratory, and Avra Synthesis Ltd. and used without further purification. The progress of the reaction was monitored by TLC using petroleum ether/ethyl acetate and chloroform/methanol as the mobile phase on pre-coated Merck TLC plates and glass plates made of F254 UV grade silica in a JSGW UV/fluorescence analysis cabinet and/or iodine chamber. Melting points were recorded on a Stuart melting point apparatus (SMP-30) with open glass capillary tubes. Infrared (IR) spectra of compounds were recorded with KBr on a Bruker FT-IR spectrophotometer. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were obtained in CDCl₃/d₆-DMSO on a Bruker Avance II (400 MHz) NMR spectrometer using TMS (δ = 0) as an internal standard at Panjab University, Chandigarh. Mass spectra were recorded on a Shimadzu GC-MS instrument (ESI), Central University of Punjab, Bathinda, Punjab, India. The purity of lead compounds was confirmed through UPLC and found to be more than 97.9%.

4.2. General procedure for the synthesis of intermediates

4.2.1. Synthesis of benzonitrile (2)

To a solution of benzaldehyde (3 mmol, 1 eq.) in NH₃ : THF (6 : 1), iodine (3 mmol, 1 eq.) was added and stirred at room temperature for 2 h. The progress of the reaction was monitored via TLC and on completion of the reaction, hydrogen peroxide was added to precipitate the product. The precipitated product was filtered under vacuum to obtain the desired intermediate (2), which was used in the next step without purification.

4.2.2. Synthesis of 2-nitrobenzonitrile (3)

To a solution of substituted benzonitrile 2 (2.5 mmol, 1 eq.) in acetic acid (3 ml), concentrated nitric acid (5 mmol, 2 eq.) was added dropwise for a period of 5 min. The reaction mixture was stirred at room temperature for 1 h. The progress of the reaction was monitored through TLC, and on completion of the reaction, crushed ice was added into the reaction mixture. The product was precipitated, filtered under vacuum and washed thoroughly with water. The solid product was dried under vacuum and was used in the next step without purification.

4.2.3. Synthesis of 2-aminobenzonitrile (4)

To a solution of substituted 2-nitrobenzonitrile (3) (2.5 mmol, 1 eq.) in ethanol (5 ml), iron (7.5 mmol, 3 eq.) and ammonium chloride (12.5 mmol, 5 eq.) were added. The reaction mixture was refluxed for 4 h to 6 h. The progress of the reaction was monitored through TLC and on completion of the reaction, the hot reaction mixture was filtered through Celite. The filtrate was concentrated under vacuum, diluted with water, and extracted with ethyl acetate (3 × 10 ml). The organic layer was washed with brine, passed through sodium sulphate, and concentrated under vacuum to afford the desired intermediate (4).

4.2.4. Synthesis of anthranilamide (5)

To a suspension of substituted 2-aminobenzonitrile (4) (2 mmol, 1 eq.) in methanol (5 ml), sodium hydroxide (2 mmol, 1 eq.) was added, and the reaction mixture was refluxed for 6 h. The progress of the reaction was monitored through TLC and on completion of the reaction, the solvent was evaporated under vacuum and the residue was diluted with water and extracted with ethyl acetate (3 × 10 ml). The combined organic layer was washed with a saturated solution of sodium bicarbonate (10 ml) and the organic layer was passed through anhydrous sodium sulphate. The organic layer was concentrated under vacuum to afford the desired anthranilamide (5), which was used in the next step without further purification.

4.2.5. Synthesis of substituted/unsubstituted quinazoline-2,4(1H,3H)-dione (6)

To a solution of anthranilamide/substituted anthranilamide (5) (1 mmol, 1 eq.) in DMSO (5 ml), urea (1.5 mmol, 1.5 eq.) and acetic acid (1 mmol, 1 eq.) were added. The reaction mixture was heated at 110 °C for 6 h to 7 h. The progress of the reaction was monitored through TLC and on completion of the reaction, heating was stopped and crushed ice was added. The product was precipitated and filtered under vacuum. The solid obtained was thoroughly washed with water and dried under vacuum. The solid precipitate (6) was used in the next step without further purification.

4.2.6. Synthesis of substituted/unsubstituted 2,4-dichloroquinazoline (7)

In a reaction vial, quinazolin-dione (6) (1 mmol, 1 eq.) was suspended in toluene (6 ml). To this reaction mixture, DIPEA (2.5 mmol, 2.5 eq.) was added and stirred for 10 min at room temperature and thereafter the reaction mixture was kept in an ice bath and POCl₃ (2.8 mmol, 2.8 eq.) was added dropwise. After addition, the reaction mixture was refluxed for 5 h. The progress of the reaction was monitored through TLC and on completion of the reaction, crushed ice was added to the reaction mixture. A saturated solution of sodium bicarbonate was used to neutralize the excess POCl₃ and thereafter the reaction mixture was extracted with ethyl acetate (15 ml × 2). The combined organic layer was washed with water and brine. Finally, the organic layer was distilled under vacuum to afford the crude product (7) which was purified through column chromatography (10% ethyl acetate in hexane).

4.2.7. General procedure for the synthesis of A4V-1 to A4V-12

To a stirred solution of 2,4-dichloroquinazoline derivative (7) (0.5 mmol, 1 eq.) in DMF (5 mL), K₂CO₃ (1.1 mmol, 2.2 eq.) was added, and the reaction mixture was stirred at room temperature for 5 min. Then, different secondary amines (benzotriazole, 1,2,4-triazole, imidazole, methylpiperazine, and phenylpiperazine) (1.1 mmol, 2.2 eq.) were added and the reaction mixture was heated at 90 °C for 3 to 4 h. The progress of the reaction was monitored through TLC and on completion of the reaction, cold water was added to the reaction mixture in order to precipitate the product which was dried under vacuum. The crude product was purified through column chromatography using ethyl acetate and hexane (in a ratio of 15–20%) as the mobile phase. All final compounds were further characterized with ¹H and ¹³C NMR and HRMS.

4.3. Spectral analysis

6,7,8-Trimethoxy-2,4-bis(4-methylpiperazin-1-yl)quinazoline (A4V-1)

White solid (0.04 g, 65%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0) δ 6.85 (s, 1H), 4.09 (s, 6H). 3.92 (s, 3H), 3.89–3.76 (m, 8H), 2.61–2.55 (m, 8H), 2.36 (s, 6H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 166.2, 154.4, 151.6, 147.0, 145.1, 111.4, 99.3, 62.3, 56.1, 54.8, 49.6, 46.1. HRMS: m/z [M + H]⁺ for C₂₁H₃₂N₆O₃, calculated 417.2614; observed 417.2615.

4,4′-(6,7,8-Trimethoxyquinazoline-2,4-diyl)dimorpholine (A4V-2)

Yellow solid (0.05 g, 73%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0) δ 6.83 (s, 1H), 4.11 (s, 3H), 4.06 (s, 3H), 3.92–3.82 (m, 15H), 3.62–3.59 (m, 4H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 165.6, 158.3, 154.4, 138.0, 137.9, 132.2, 129.2, 128.3, 128.2, 127.2, 127.1, 125.1, 112.0, 63.2, 53.2, 49.8, 43.9. HRMS: m/z [M + H]⁺ for C₁₉H₂₆N₄O₅, calculated 391.1981; observed 391.1985.

2,4-Di(1H-imidazol-1-yl)-6,7,8-trimethoxyquinazoline (A4V-3)

Brown solid (0.05 g, 74%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): δ 8.29 (s, 1H), 8.00 (d, J = 4 Hz, 2H), 7.67 (d, J = 4 Hz, 2H), 7.36 (s, 1H), 7.14 (s, 1H), 4.20 (s, 6H), 3.96 (s, 3H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 154.9, 153.9, 147.0, 146.3, 137.4, 136.3, 131.1, 130.8, 119.4, 116.8, 112.8, 97.7, 62.7, 61.6, 56.5. HRMS: m/z [M + H]⁺ for C₁₇H₁₆N₆O₃, calculated 353.1362; observed 353.1369.

6,7,8-Trimethoxy-2,4-di(4H-1,2,4-triazol-4-yl)quinazoline (A4V-4)

Yellow solid (0.03 g, 61%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): δ 8.85 (s, 2H), 8.81 (s, 2H), 7.15 (s, 1H), 4.14 (s, 6H), 3.91 (s, 3H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 155.2, 154.1, 153.7, 147.7, 142.3, 137.6, 136.5, 131.3, 131.0, 97.9, 62.9, 62.0, 56.7. HRMS: m/z [M + H]⁺ for C₁₅H₁₄N₈O₃, calculated 355.1267; observed 355.1260.

2,4-Bis(1H-benzo[d][1,2,3]triazol-1-yl)-6,7,8-trimethoxyquinazoline (A4V-5)

White solid (0.04 g, 77%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): δ 8.76 (s, 1H), 8.50 (d, J = 8 Hz, 1H), 8.44 (d, J = 8 Hz, 1H), 8.31–8.28 (m, 2H), 7.91–7.84 (m, 2H), 7.79–7.73 (m, 2H) 4.18 (s, 6H), 3.95 (s, 3H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 165.3, 149.5, 148.3, 148.2, 145.7, 144.8, 143.2, 140.5, 137.4, 136.4, 128.4, 127.6, 121.4, 116.9, 113.0, 112.0, 106.0, 102.1, 61.1, 57.2. HRMS: m/z [M + H]⁺ for C₂₃H₁₈N₄O₃, calculated 455.1580; observed 455.1585.

2,4-Bis(1H-benzo[d][1,2,3]triazol-1-yl)quinazoline (A4V-6)

Brown solid (0.06 g, 81%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): δ 9.46 (d, J = 8 Hz, 1H), 9.06 (d, J = 4 Hz, 1H). 8.79 (d, J = 8 Hz, 1H), 8.26–8.19 (m, 3H), 8.07–8.03 (m, 1H), 7.81 (d, J = 8 Hz, 2H), 7.68 (t, J = 8 Hz, 1H), 7.62–7.59 (m, 1H), 7.55–7.51 (m, 1H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 156.9, 154.3, 151.5, 146.9, 145.9, 135.7, 132.9, 131.8, 130.8, 129.5, 128.8, 128.7, 128.5, 126.4, 125.5, 120.3, 120.3, 120.3, 116.3, 115.8. HRMS: m/z [M + H]⁺ for C₂₀H₁₂N₈ calculated 365.1263; observed 365.1262.

2,4-Bis(1H-imidazol-1-yl)quinazoline (A4V-7)

Yellow solid (0.05 g, 64%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): δ 8.51 (d, J = 8 Hz, 1H), 8.12 (d, J = 8 Hz, 1H), 7.87 (s, 1H), 7.80 (s, 1H), 7.70 (t, J = 8 Hz, 1H), 7.64–7.53 (m, 3H), 7.31 (d, J = 8 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 156.0, 149.1, 137.3, 134.8, 132.5, 131.1, 129.3, 128.4, 127.3, 125.5, 122.8, 121.4, 116.0. HRMS: m/z [M + H]⁺ for C₁₄H₁₀N₆ calculated 263.1045; observed 263.1041.

2,4-Bis(4H-1,2,4-triazol-4-yl)quinazoline (A4V-8)

Pale yellow solid (0.04 g, 67%); ¹H NMR (CDCl₃, 400 MHZ, δ with TMS = 0): δ 8.85 (s, 2H), 8.81 (s, 2H), 8.59 (d, J = 4 Hz, 1H), 8.33 (d, J = 8 Hz, 1H), 7.84–7.80 (m, 1H), 7.75–7.71 (m, 1H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 159.4, 154.4, 147.8, 146.9, 143.4, 133.5, 129.4, 128.3, 126.5, 116.3. HRMS: m/z [M + H]⁺ for C₁₂H₈N₈, calculated 265.0950; observed 265.0951.

4,4′-(Quinazoline-2,4-diyl)dimorpholine (A4V-9)

Yellow solid (0.05 g, 74%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0) δ 7.77 (d, J = 8 Hz, 1H), 7.67 (d, J = 8 Hz, 1H), 7.55–7.51 (m, 1H), 7.32–7.28 (m, 1H), 3.82–3.72 (m, 16H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 165.6, 158.3, 154.4, 138.0, 137.9, 132.2, 129.2, 128.3, 128.2, 127.2, 127.1, 125.1, 112.0, 63.2, 53.2, 49.8, 43.9. HRMS: m/z [M + H]⁺ for C₁₆H₂₀N₄O₂, calculated 301.1665; observed 301.1661.

2,4-Bis(4-benzylpiperazin-1-yl)quinazoline (A4V-10)

White solid (0.035 g, 60%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): δ 7.66 (d, J = 8 Hz, 1H), 7.48 (d, J = 4 Hz, 2H). 7.36–7.31 (m, 8H), 7.28–7.25 (m, 2H), 7.05–7.02 (m, 1H), 3.90 (s, 4H), 3.66 (s, 4H), 3.56 (d, J = 8 Hz, 4H), 2.61 (s, 4H), 2.51 (s, 4H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 165.6, 158.3, 154.4, 138.0, 137.9, 132.2, 129.2, 129.2, 128.3, 128.2, 127.2, 127.1, 125.1, 112.0, 63.2, 53.2, 49.8, 43.9. HRMS: m/z [M + H]⁺ for C₃₀H₃₄N₆ calculated 479.2923; observed 479.2920.

2,4-Bis(4-phenylpiperazin-1-yl)quinazoline (A4V-11)

Brown solid (0.040 g, 68%); ¹H NMR (CDCl₃, 400 MHz, δ with TMS = 0): δ 7.76 (d, J = 8 Hz, 1H), 7.59 (d, J = 4 Hz, 2H). 7.46–7.41 (m, 8H), 7.37 (d, J = 8 Hz, 1H), 7.35 (s, 1H), 7.14–7.10 (m, 1H), 4.01 (s, 4H), 3.76 (s, 4H), 2.73 (s, 4H), 2.61 (s, 4H). ¹³C NMR (100 MHz, CDCl₃, TMS = 0): 155.2, 154.1, 137.6, 136.5, 131.3, 131.0, 97.9, 62.9, 62.0, 56.7. HRMS: m/z [M + H]⁺ for C₂₈H₃₀N₆ calculated 451.2610; observed 451.2615.

2,4-Bis(4-methylpiperazin-1-yl)quinazoline (A4V-12)

Yellow solid (0.04 g, 54%); ¹H NMR (CDCl₃, 400 MHZ, δ with TMS = 0): δ 7.67 (d, J = 8 Hz, 1H), 7.51–7.47 (m, 2H), 7.05–6.98 (m, 1H), 3.93–3.89 (m, 4H), 3.70 (t, J = 4 Hz, 4H), 2.61 (t, J = 4 Hz, 4H), 2.51 (t, J = 4 Hz, 4H), 2.37 (s, 6H). ¹³C-NMR (100 MHz, CDCl₃, TMS = 0): 165.8, 158.4, 154.6, 132.6, 126.4, 125.3, 120.8, 112.2, 55.3, 55.1, 49.9, 46.4, 46.3, 44.1. HRMS: m/z [M + H]⁺ for C₁₈H₂₆N₆ calculated 327.2297; observed 327.2292.

4.4. Biological studies

4.4.1. Cell culture and in vitro cell cytotoxicity assay

The synthesized compounds were assayed for their cytotoxicity by in vitro cell viability assay on different types of cancer cell lines (MCF-7, HCT-116, and SHSY-5Y) and on normal cell line (HEK-293 T). All cell lines (MCF-7, HCT-116, SHSY-5Y and HEK-293 T) were procured from the cell repository at the National Centre for Cell Science, Pune (Maharashtra), India, and cultured in Dulbecco's modified Eagle medium (DMEM, Gibco™) additionally supplemented with 10% fetal bovine serum (Gibco™) and 1% HEPES (Invitrogen, Life Technologies™), while 1% sodium pyruvate (Invitrogen, Life Technologies™) was additionally supplemented to the MCF-7 cell line and all the cell lines were maintained at 37 °C in a CO₂ (5%) incubator. For viability assays, cells were seeded in 96-well plates at a density of 2 × 10⁴ cells per well and further incubated for the next 12 hours to allow cell adherence to the substratum. Stock solutions of all the compounds were prepared in DMSO (HiMedia) and final working concentrations (1 μM, 5 μM, 10 μM and 25 μM) were prepared in tissue culture medium and used for treatment of the cells for the next 48 hours. Only DMSO was used as a vehicle control for all the drugs. After 48 h, 10 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Aldrich) reagent (5 mg ml⁻¹) was added to each well and incubated at 37 °C in a CO₂ incubator for the next 2 h as described earlier.³⁷ MTT dye is reduced by succinate dehydrogenase from live and healthy cells which converts it into insoluble purple formazan crystals. After the incubation all the medium was aspirated and 100 μl of DMSO (Sigma Aldrich) was used to uniformly dissolve the formazan crystals using an orbital shaker at 250 RPM for 30 min. Absorbance was recorded at 570 nm on a multimode microplate reader (Infinite M200 Pro, Tecan Switzerland). Cell viability was calculated against the control cells without any compound.

4.4.2. Annexin V/PI apoptosis assay

Actively growing cancer cells (SHSY-5Y) were seeded at a density of 2 × 10⁵ in a 12-well plate and treated with different concentrations of the most potent compounds A4V-3 and A4V-5. Cells were incubated for the next 48 h and colchicine was used as positive control. Additionally, sets like negative and single stain controls were also prepared to set the voltage and fluorescence compensation. After 48 h of incubation, these cells were trypsinized, washed with 1× PBS, centrifuged, and the pellet was resuspended in fluorescence-activated cell sorter (FACS) binding buffer. Cells were then stained using an FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™, Lot. 7303808) as per the manufacturer's protocol. Post incubation, the fluorescence intensity the cells were measured using an LSRFortessa cell analyzer. 10 000 events per sample were taken during data acquisition and data analysis was done using in-built BD FACSDiva software.³⁸

4.4.3. Cell cycle analysis

Healthy cells (SHSY-5Y) were seeded at a density of 2 × 10⁵ cells per well in a 12-well plate and kept at 37 °C in a CO₂ incubator overnight to allow cell adherence. After that, cells were treated with different concentrations of the test compounds A4V-3 and A4V-5 and incubated for the next 48 h. Post incubation cells were trypsinized using warm trypsin and washed once with ice-cold 1× PBS and centrifuged. Then, the cells were resuspended in chilled 70% ethanol for the next 3 h followed by centrifugation and washing of cells with ice-cold PBS and then stained with 200 μl of 40 μg mL⁻¹ propidium iodide (BD Pharmingen™) as described earlier.³⁹ Cells were then analyzed using the LSRFortessa cell analyzer. 10 000 events per sample were taken during data acquisition and data analysis was done using in-built BD FACSDiva software.

4.4.4. JC-1 assay

SHSY-5Y cells were seeded at a density of 2 × 10⁵ cells per well in a 12-well plate and kept at 37 °C in a CO₂ incubator overnight. Cells were then treated with different concentrations of the test compounds A4V-3 and A4V-5 for the next 48 h. Post treatment cells were harvested, washed once with 1× PBS, centrifuged and again resuspended in fresh 1× PBS. After that 2.5 μM JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide, BD Sciences) was added to all the samples and then incubated in the dark at 37 °C under gentle shaking for the next 15 min.⁴⁰ Cells were then analysed using Synergy H1 microplate reader green monomers with Ex 485 nm/Em 535 nm and red J aggregate with Ex 560 nm/Em 595 nm.

4.4.5. Tubulin polymerization assay

Tubulin polymerization was determined using a fluorescence-based tubulin polymerization assay kit (Cytoskeleton catalog no. BK011P, Cytoskeleton Tubulin isolated from porcine brain tissue was used in this commercial kit) according to the manufacturer's protocol.⁴¹ It is based on the principle that light is scattered by microtubules to an extent that is proportional to the concentration of the microtubule polymer. Polymerization of tubulin will alter as the compounds interact with tubulin, and this altered tubulin polymerization was detected using a spectrophotometer. Tubulin was resuspended in ice-cold tubulin buffer (80 mM PIPES, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP, 20% (v/v) glycerol) and added to the wells of a 96-well plate containing the designated concentration of the drug or vehicle. The samples were mixed well, and tubulin assembly was monitored at 1 min intervals for 60 min at 37 °C using a plate reader (Victor3V, PerkinElmer Life Sciences).

4.5. Computational studies

4.5.1. Docking studies

Docking study was performed to analyse the mode of interaction between synthesized ligand A4V-3 and tubulin protein. For the generation of 2D and 3D structures of synthesized ligands, ChemBioDraw Ultra 15 and Maestro 12.3 (Schrödinger) software were used. The X-ray crystal structure of tubulin (PDB ID 1JFF) stabilized with taxol was downloaded from the Protein Data Bank (https://www.rcsb.org).⁴² All the synthesized compounds and protein were separately prepared using LigPrep and Protein Preparation Wizard. All the different possible conformations of ligands were generated, and energy was minimised by the LigPrep module. Similarly, protein was also prepared by adding missing polar hydrogens and side chains that are missed in protein by Protein Preparation Wizard. It also removes all the water molecules that are present other than the active site cavity. The prepared protein was energy minimized using OPLS2005 force field. After ligand and protein preparation, a grid was generated using the Receptor Grid Generation Panel at the site in the protein where the ligand is to be docked by replacing the crystalized ligand. All atoms within 5 Å around the co-crystallized ligand in the crystal coordinates of tubulin were chosen as binding sites. For each compound, the top-score docking poses were chosen for final ligand-target interaction analysis employing the XP interaction visualizer of Maestro 12.3 software. The QikProp application of Schrödinger suite was utilised for the determination of drug-like and ADME properties of the compounds.⁴³

4.5.2. Molecular dynamics simulation

MD simulation study of the lead compound was performed to investigate the interactions of the protein and ligand complex for 100 ns via GROMACS 2021.3, carried out as previously reported by Jangid K. et al.⁴⁴ The CHARMM36-Mar2019 force field was utilized for the generation of complex topology, and the TIP3P solvation model used to solvate the system. A water box was created with dimensions at a minimum distance of 1.5 nm between the protein surface and box boundaries. To neutralize the ligand–protein complex system, appropriate amounts of Na⁺ and Cl⁻ were added as counter ions. The system was minimized through three successive steps of energy minimization and NVT and NPT equilibration. The energy was minimized via the steepest descent algorithm, while NVT equilibration for protein was run for 500 ps at 310 K with the use of a Nose–Hoover thermostat (coupling constant = 0.1 ps).⁴⁵ Next, NPT equilibration was done at 500 ps (pressure = 1 bar, coupling constant = 1 ps) utilizing the Parrinello–Rahman algorithm.⁴⁶ The next production run was carried out for 100 ns. During the simulation process the time step was fixed at 2 fs. Constraints of bond length were calculated using LINear Constraint Solver (LINCS) algorithm.⁴⁷ Along with that PME (particle mesh Ewald) strategy having 0.16 nm grid spacing and 1.2 nm cut-off radius was employed at 1 nm van der Waals cut-off distance. For analysis of the MD simulation trajectory, GROMACS analytic tools, including RMSD (root mean square deviation), RMSF (root mean square fluctuation), rGy (radius of gyration) and hbond (hydrogen bonding) were computed.^48,49

4.5.3. MM-GBSA calculations

Binding free-energy calculations for A4V-3 with 1JFF were done using the Prime module of Schrödinger suite 2021. The binding free energy of the 1JFF–ligand complex was calculated using the prime energy, a molecular mechanics + implicit solvent energy (kcal mol⁻¹).

MMGBSA dG Bind = PrimeEnergy(Optimized Complex) − PrimeEnergy(Optimized Free Ligand) − PrimeEnergy(Optimized Free Receptor).

Abbreviations

TBA

Tubulin binding agent

MSA

Microtubule-stabilizing agent

MDA

Microtubule-destabilizing agent

MD

Molecular dynamics

ADME

Absorption distribution metabolism and excretion

log P

Partition coefficient of a molecule between an aqueous and a lipophilic phase (octanol and water)

log S

Aqueous solubility

#metab

Number of likely metabolic reactions

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare no potential conflict of interest.