Discovery of sulfonamide-tethered isatin derivatives as novel anticancer agents and VEGFR-2 inhibitors

Abstract

In this work, new isatin-based sulphonamides (6a-i, 11a-c, 12a-c) were designed and synthesised as potential dual VEGFR-2 and carbonic anhydrase inhibitors with anticancer activities. Firstly, all target isatins were examined for in vitro antitumor action on NCI-USA panel (58 tumour cell lines). Then, the most potent derivatives were examined for the potential CA inhibitory action towards the physiologically relevant hCA isoforms I, II, and tumour-linked hCA IX isoform, in addition, the VEGFR-2 inhibitory activity was evaluated. The target sulphonamides failed to inhibit the CA isoforms that could be attributable to the steric effect of the neighbouring methoxy group, whereas they displayed potent VEGFR-2 inhibitory effect. Following that, isatins 11b and 12b were tested for their influence on the cell cycle disturbance, and towards the apoptotic potential. Finally, detailed molecular modelling analyses, including docking and molecular dynamics, were carried out to assess the binding mode and stability of target isatins.

Keywords:

• Synthesis
• antitumor agents
• biological activities
• VEGFR-2 inhibitors
• molecular dynamics

Introduction

Cancer, a condition of uncontrolled cell growth, remains the most difficult life-threatening disease to treat, despite advances in understanding of its biochemistry and progression¹. The rising prevalence of cancer treatment failure derives mainly from antitumor drug resistance in cancer cell, posing new challenges to the healthcare system². Many targets have been identified that are involved in one or more steps in regulating tumour cell growth or death³. Combining anticancer drugs is becoming a widely accepted strategy and treatment standard for avoiding drug resistance and treatment failure⁴.

Carbonic anhydrase (CA) is an effector enzyme in the tumour cell survival mechanism that regulates the pH of the tumour microenvironment⁵. CAs are zinc metalloenzymes that catalyse the reversible interconversion of CO₂ and bicarbonate ions. Several CA isoforms have been identified, with CA IX and CA XII isoforms being upregulated in nearly all hypoxic tumours, promoting tumour growth and metastasis. CA IX isoform overexpression is associated with a poor prognosis in many cancers and is involved in cell proliferation and communication⁶. The primary sulfonamide-based small molecules were discovered to be the most potent chemotype of the identified CA inhibitors (CAIs)⁷. Among those sulfonamide-based inhibitors, SLC-0111 (Figure 1) displayed effective CA IX and XII inhibitory action, and is currently being examined in the clinical trials for the hypoxic malignancies management⁸.

Figure 1. Structure of Sunitinib, SLC-0111, WEG-104, compounds I-II, and target compounds 6a-i, 11a-c and 12a-c.

The clinically validated anticancer drug target, vascular endothelial growth factor receptor-2 (VEGFR-2) is one of the receptor tyrosine kinases that have critical role in vasculogenesis and angiogenesis in many solid cancers. VEGFR-2 mediates the phosphorylation of many proteins in the downstream signalling pathways promoting tumour angiogenesis⁹. Clinical antiangiogenic drugs that are VEGFR-2 inhibitors, such as Sunitinib (Figure 1) showed to achieve normal tumour vasculature and, consequently, contribute in improving chemotherapy treatment. These treatments are planned to hit both antiapoptotic functions and pro-angiogenic activities of VEGF.

Isatin, an endogenic molecule in mammalaian tissues including human, stands for valuable privileged scaffold in drug design and pharmaceutical chemistry¹⁰. Isatin-tethered compounds have been found to display various pharmacological effects, in particular carbonic anhydrase^11–14 and VEGFR-2^15–17 inhibitory activities. In the last few years, isatin motif has been exploited to develop several CAIs with effective in vitro and in vivo antitumor activities, such as WEG-104^18,19 and compound I²⁰ (Figure 1). In 2019, we reported a new set of N-substituted isatin derivatives as promising CAIs. Among this set, compound II (Figure 1) displayed excellent activity against cancer-related CA IX and XII isoforms; K_I = 5.2 and 6.3 nM, respectively. Also, it exerted good VEGFR-2 inhibitory action (IC₅₀ = 260.64 nM), as well as effective cell growth inhibitory action on breast cancer cell lines²¹.

Herein, we decided to develop new isatin-based sulphonamides (6a-i, 11a-c and 12a-c) as potential dual VEGFR-2 and carbonic anhydrase activities. All target synthesised isatin derivatives 6a-i, 11a-c and 12a-c will be evaluated for in vitro antitumor action on NCI-USA panel covering 58 distinct human tumour cell lines. Thereafter, the most potent derivatives will be examined for the potential CA inhibitory action towards physiologically relevant hCA isoforms I, II, and cancer-related hCA IX isoform, in addition, the inhibitory activity against VEGFR-2 will be evaluated.

Results and discussion

Chemistry

General preparation procedures used in synthesising the designed compounds 6a-i are shown in Scheme 1. The first step in synthesis was the preparation of benzenesulfonyl chloride 2 using thionyl chloride and chlorosulfonic acid in performing chlorosulfonation of compound 1. An excess chlorinating agent 2:1 was used to enhance the yield after 26 h. The formation of benzenesulfonamide intermediate 3 was accomplished by reacting compound 2 with ammonia using ethanol as solvent²². The synthesis of hydrazone intermediate 4 was carried out by refluxing compound 3 with hydrazine hydrate for 4 h in the presence of glacial acetic acid as catalyst and ethanol as solvent²³. The target compounds 6a-i were prepared by reacting them with various isatin derivatives 5a-i in the presence of a catalytic amount of glacial acetic acid and under reflux conditions^24–26.

Scheme 1. General procedure for the synthesis of target compounds 6a-i. Reagents and conditions: i) HOSO₂Cl, SOCl₂ at 0 °C and then rt, 26 h ii) EtOH, ammonia, rt iii) NH₂NH₂.H₂O, AcOH (cat.), and EtOH, reflux, 4 h. iv) EtOH, AcOH (cat.), reflux, 6–8 h.

The general procedures for synthesising target N-methylated/benzylated isatin derivatives are illustrated in Scheme 2. The N-methylation step of isatins 5a, 5d, and 5f was carried out by their reaction with methyl iodide 7 in the presence of potassium carbonate and acetonitrile to afford desired N-methylated isatins 9a-c, which undergo a further condensation reaction with compound 4 in glacial acetic acid to obtain the final compounds 11a-c. Similarly, benzylated isatin derivatives 10a-c were synthesised by reacting isatins 5a, 5d, and 5f with benzyl bromide 8 in the presence of acetonitrile and potassium carbonate under refluxing conditions²⁴. Thereafter, N-benzyl derivatives 10a-c were condensed with hydrazone intermediate 4 using glacial acetic acid as a catalyst to produce target 2-indolinones 12a-c.

Scheme 2. General procedure for the synthesis of target compounds 11a-c and 12a-c. Reagents and conditions: i) K₂CO₃, acetonitrile, reflux, 5 h; ii) EtOH, AcOH, reflux, 4 h.

All the newly prepared molecules were confirmed with ¹H and ¹³C NMR, mass spectroscopy, and elemental analysis. The protons and carbons signals emerged in the expected chemical shifts (experimental section). According to the ¹H NMR spectra, the target isatin derivatives exist as E- and Z-isomers, but they interconvert fast in solution at room temperature and cannot be separated. As previously described, the ratio of isomers for isatin hydrazones is solvent and temperature dependent^27–29, we have reported several studies with such E/Z mixture for different isatin hydrazones^24,30–32.

Biological activity

In vitro single-dose cellular antiproliferative assay

The designed isatin derivatives 6a-i, 11a-c and 12a-c were examined for in vitro antitumor activity on NCI panel involving 58 distinct human tumour cell lines covering nine distinct types of cancer, at single concentration of 10 μM (supplementary file). For analysing the activity of the new isatin series on 58-cancer cell line, GI (mean % growth inhibition) displayed by 6a-i, 11a-c and 12a-c on different cell lines were calculated, and results were presented in Figure 2. Based on the findings, we concluded that none of compounds 6a, 6c, or 11a were able to increase cell inhibition by more than 20%. And by looking at the mean growth inhibition of the other compounds on each cancer, we discovered that, in addition to CNS cancer in compound 11c and leukaemia in compounds 12b and 12c, all the compounds had the strongest mean growth inhibition on breast cancer cell lines. We also found that the most sensitive cells in breast cancer subpanel is the T47D cells, and it responded best GI% to compounds 6f, 6i, 11b, 11c, 12a, 12b, and 12c with GI%; 54, 32, 55, 52, 57, 28 and 39, respectively (Figure 2).

Figure 2. % GI for the highly affected cell lines on treatment by the target isatin derivatives using single dose of 10 μM.

In vitro cytotoxicity against T47D

For further exploration of possible anti-proliferative properties of isatin derivatives, dose-response evaluation was carried out on the T47D breast cancer cells (the most sensitive cancer cell line according to % GI in vitro single-dose cellular antiproliferative assay), employing the sulforhodamine B colorimetric test. Doxorubicin was set as reference in this assay. IC₅₀ values were ascertained and are displayed in Table 1. Derivatives 6f, 11b-c, and 12b revealed good cytotoxic action demonstrating IC₅₀ from 1.83 to 10.40 µM in comparison to doxorubicin (IC₅₀ of 2.26 µM). As earlier noted in single-dose assay, a bromo-isatin analogue of C-5-functionalized isatins was preferred in the N-(un)alkylated derivatives of isatin series. Compound 6f (IC₅₀ = 5.45 ± 0.24 µM) was found to be the most potent among the studied series. Regarding the N-alkylated/arylated analogs of isatin series with methyl or benzyl moiety, the presence of a chloro substitution at position 5 led to the best potency (N-methyl analogue 11b, IC₅₀ of 1.83 ± 0.08 µM and N-benzyl analogue 12b, IC₅₀ of 3.59 ± 0.16 µM), followed by the C-5-bromo substituted derivatives (compounds 11c and 12c, IC₅₀ of 10.40 ± 0.47 and 16.52 ± 0.74 µM respectively) as shown in Table 1.

Table 1. Cytotoxicity of selected compounds 6f, 6i, 11b-c, and 12a-c (IC₅₀) against T47D cells.

Compound	IC50 ± S.D. (µM) T47D^a
6f	5.45 ± 0.24
6i	24.13 ± 1.08
11b	1.83 ± 0.08
11c	10.40 ± 0.47
12a	11.58 ± 0.52
12b	3.59 ± 0.16
12c	16.52 ± 0.74
Doxorubicin^33	2.26 ± 0.10

^aIC₅₀ values are mean ± SD of triplicate experiments.

In vitro VEGFR-2 and carbonic anhydrase inhibition activity

Isatins 6f, 11b-c, and 12b, which revealed the most cytotoxic activity on the T47D cells that characterised by the overexpression of VEGFR-2 and carbonic anhydrase^34–36, were selected for further analysis of their in vitro VEGFR-2 and CA inhibition properties (Table 2).

Table 2. IC₅₀ values of selected compounds 6f, 11b-c, and 12b against VEGFR-2 and CA inhibition activity.

Compound	VEGFR-2^a		Carbonic anhydrase activity KI (µM)^b
	% Inhibition at 10 µM	IC50 ± S.D. (nM)	CA I	CA II	CA IX
6f	86.82	56.70 ± 0.72^a	>100	>100	>100
11b	72.67	63.40 ± 0.72^a	>100	>100	>100
11c	92.03	30.10 ± 0.31^a	>100	>100	>100
12b	97.18	23.10 ± 0.41^a	>100	>100	>100
Sorafenib	96.40	29.70 ± 0.17^a	–	–	–
Acetazolamide (AAZ)	–	–	0.25	0.012	0.026

^aIC₅₀ value is mean ± SD of triplicate experiments, ^bMean of triplicate different assays, via stopped flow assay.

All derivatives under test demonstrated good VEGFR-2 inhibition with an IC₅₀ of 23.10 to 63.40 nM, with compound 12b being the most potent (IC₅₀ = 23.10 nM) and compound 11c being equivalent to the commonly used kinase inhibitor sorafenib (IC₅₀ = 30.10 nM), which had an IC₅₀ value of 29.70 nM and the remaining two being marginally less potent.

On the other hand, all the evaluated isatin derivatives failed to inhibit the CA isoforms (K_I > 100 µM), contrary to predictions, that could be attributable to the steric hindrance by the neighbouring methoxy group³⁷.

Cell cycle distribution analysis

To learn more about their cellular mechanisms of action, the most potent compounds (11b and 12b) were selected. DMSO (control) or compounds 11b and 12b were applied to T47D cells, and flow cytometry was used to determine the DNA concentration. Figure 3 presents the findings. Both compounds showed different cell-cycle arrest patterns.

Figure 3. Phases distribution of T47D cells upon incubation with 11b and 12b.

Cells treated with 11b revealed an increase in sub-G1 and G0/G1 phases (45.88% and 68.42%, respectively), compared to 61.39% and 2.41%, respectively, in control. Additionally, compound 12b increased the proportion of cells in the S and sub-G1 phases of the cell cycle from 28.55% and 2.41% for untreated T47D cells to 41.05% and 39.15%, respectively (Figure 3).

Apoptosis assay

To explore link between growth suppression activity of compounds 11b and 12b with initiation of apoptosis indicated by rising population of sub-G1 in treated T47D cell lines, the Annexin V-FITC/PI double labelling (AV/PI) apoptosis assay was done. The outcomes of the experiment are shown in Figure 4, showing that compounds 11b and 12b caused T47D cell lines to undergo early and late apoptosis. In fact, % of apoptotic cells amplified from 0.41% at early and 0.22% at late apoptosis in T47D cells that had not been treated to 31.0% of early and 12.07% of late apoptosis in T47D cell that had been treated with compound 11b and to 23.5% at early and 9.4% at late apoptosis of T47D cells that had been treated with compound 12b. According to these findings, compounds 11b and 12b increased the overall apoptosis of T47D cells by roughly 19 and 16.2 times, respectively, in comparison to the control.

Figure 4. Apoptosis rate quantification (%) and necrosis in (A) flow cytometry, effect of (B) control, (C) 11b and (D) 12b on annexin V-FITC-positive staining (%) in T47D cell line.

In silico studies

Molecular docking

The VEGFR-2 crystal structure (code 4ASD) was used in the molecular docking investigation that was done for predicting binding mode of 12b as most active compound presented in this study. As predicted, 12b fits deeply into the active site region with nearly the same orientation as the co-crystallized ligand (sorafenib, Figure S64).

In addition, the H-bonds were formed by the sulfamoyl amino group with Ile1025 and His1026 and between the isatin carbonyl group with Asp1046 (Figure 5). The heterocyclic rings of 12b were involved in π-ion interactions with Asp1046, Lys868 and Glu885 while Cys1045 interacted with the five membered ring of isatin via π-sulfur interaction. Additionally, the benzyl ring and other hydrophobic components of 12b were shown to interact with several hydrophobic residues found in the active site of the VEGFR-2 receptor, including Val848, Ala866, Leu889, Val899, Leu901, Val914, Val916, and Leu1035. All the aforementioned interactions were responsible for the good affinity of 12b to the studied receptor as indicated by the docking score of −8.5 kcal/mol.

Figure 5. Docking of 12b inside the active site of VEGFR-2 receptor (code: 4ASD); active site view (top) and 2D schematic view of the interactions (bottom).

Molecular dynamic simulation

After the docking process of 12b into the VEGFR-2 receptor, the stability of the complex structure needed to be examined. To test the durability of the best pose for the 12b-VEGFR-2 complex at room temperature circumstances, a 100 ns MD simulation was used. Over this simulation period, the complex system’s temperature, pressure and potential energy, are shown in Figure 6 showing converged system throughout whole simulation period.

Figure 6. From left to right: (A) Temperature, (B) pressure and (C) potential energy during the 100 ns MD simulations.

Analysing trajectories after simulation showing 12b ligands stayed attached to active site in protein pocket as indicated by the calculated RMSD, radius of gyration and average of distance at mass centre between ligand and protein Figure 7.

Figure 7. RMSD, Radius of gyration and average centre of mass distance of heavy atoms of 12b during 100 ns MD simulation.

Furthermore, SASA and RMSF of protein over the simulation period showed no significate change in absence and presence of 12b as shown in Figure 8.

Figure 8. RMSF and SASA of VEGFR-2 in presence and absence of 12b over 100 ns MD simulation.

Evaluating interactions of 12b to the nearby residues within the active site of VEGFR-2 showed various stable interactions over the simulation period (Figure 9). Different forces contributing to the affinity of 12b to the receptor were observed during 100 ns of the simulation run including π-stacking, H-bonding and hydrophobic, Figure S65–67. Figure 10 depicts the total number of H-bonds formed by the protein and ligand during the simulation period. On average, there were two to three H-bonds to the VEGFR-2 receptor remained stable throughout the entire simulation. Finally, all these interactions were reflected in the binding free energy (MMPBSA) that were carried out to assess the stability of the formed complex structure (Table 3).

Figure 9. Different types of interaction exhibited by 12b with the amino acids within the active site of VEGFR-2 during the whole MD simulation frames.

Figure 10. Hydrogen bonds between VEGFR-2 and 12b during 100 ns MD simulation.

Table 3. Free binding energies of 12b with VEGFR-2 in kJ/mol.

Binding energies	kJ/mol
Electrostatic energy	−84.723 ± 46.032
Polar solvation energy	189.645 ± 48.831
SASA energy	−24.848 ± 1.635
van der Waal energy	−195.211 ± 25.865
$\Delta G$	−115.137 ± 28.682

Conclusions

In the current study, different sets of isatin-based sulphonamides (6a-i, 11a-c and 12a-c) were reported with the prime goal of developing new anticancer candidates with dual VEGFR-2 and carbonic anhydrase inhibitory actions. In vitro anticancer activities of target isatins were first explored towards 58 tumour cell lines (NCI-USA panel). The findings revealed that the breast cancer subpanel was the most susceptible to the influence of target isatins. In particular, the T47D cells growth was effectively inhibited by compounds 6f, 6i, 11b-c and 12a-c. Then, the IC₅₀ values of these isatins towards T47D cells were determined (IC₅₀ range: 1.83 − 24.13 µM). Thereafter, the VEGFR-2 and carbonic anhydrase inhibitory actions of isatins 6f, 11b-c, and 12b were evaluated. While the target isatin sulphonamides potently inhibited VEGFR-2 (IC₅₀ range: 23.10 − 63.40 nM), they failed to inhibit the CA isoforms (K_I > 100 µM), contrary to predictions, that could be attributable to the steric hindrance by the neighbouring methoxy group. Moreover, isatins 11b and 12b were tested for their effect on cell cycle, and towards apoptotic potential.

Experimental

Chemistry

General

Reactions progress was checked by TLC sheets (Silica 60 F₂₅₄, Fastman Kodak Co.) using methanol and chloroform: (10: 90) as elution system: and visualisation was done at 254 nm. Melting point was measured on Stuart SMP10 using the open capillary technique and was reported uncorrected. ¹³C NMR (101–126 MHz) and ¹H (400–500 MHz) spectra were obtained using Bruker FTNMR spectrometer using DMSO-d₆ as a solvent. Spectra of high-resolution mass were recorded on Bruker MicroTOF spectrometer. Elemental analysis (% C, H, N, and S) was done using CHNS analyser (2400 Perkin–Elmer). The spectral data and their interpretation were deposited in the supplementary file.

General steps for preparation of 5-acetyl-2-methoxybenzene-1-sulfonyl chloride (2)

Chlorosulfonic acid (90 mmol) was stirred with thionyl chloride (30 mmol) in an ice bath for 30 min. After that, 4-methoxyacetophenone 1 (15 mmol) was added to the mixture dropwise, followed by stirring the reaction mixture for 26 h at room temperature. Subsequently, ice water was added, and the resulting pale orange precipitate 2 was taken after being filtered and washed using H₂O (3.35 g, 90%), Mp: 102–103 °C^22,37.

General steps for preparation of 5-acetyl-2-methoxybenzenesulfonamide (3)

Compound 2 (10 mmol) was stirred in ethanol (30 ml) as solvent at room temperature while 30 ml ammonia was added to the mixture. Completion of the reaction detected via TLC. The pink precipitated compound 3 was isolated and purified using ethanol and water (1.81 g, 79%), Mp: 202–203 °C^22,38.

General procedure for preparation of 5-[(1E)-ethanehydrazonoyl]-2-methoxybenzene-1-sulfonamide (4)

Compound 3 (7.9 mmol) was refluxed with hydrazine hydrate (10 mmol), glacial acetic acid (2.5 ml) and ethanol (50 ml) for 4 h. Next, the mixture brought to room temperature, then its extraction with DCM (50 ml × 3). The obtained product 4 was dried over Na₂SO₄, concentrated under vacuum, and used in the next step without further purification.

General steps for preparation of 2-methoxy-6-[1–(2-[2-oxo-2,3-dihydro-1H-indol-3-ylidene]hydrazin-1-ylidene)ethyl]benzene-1-sulfonamide derivatives (6a-i)

Compound 4 (0.4 mmol) was stirred with different derivatives of isatin 5a-i (0.4 mmol) using absolute ethanol as solvent under reflux conditions for 6–8 h with the addition of glacial acetic acid using catalytic amount. The formed precipitate was filtered and washed with ethanol before recrystallizing it from DMF to afford desired compounds 6a-i.

General steps for preparation of N-methylated derivatives of 2-methoxy-6-[(1E)-1–(2-[(3E)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]hydrazin-1-ylidene)ethyl]benzene-1-sulphonamides (11a-c)

Isatin derivatives 5a, 5d and 5f (2 mmol) were refluxed with (2.8 mmol) of methyl iodide 7 in acetonitrile (20 ml) using a catalytic amount of potassium iodide and dry potassium carbonate (10 mmol). TLC was used in monitoring the reaction progression. After reaction completion, it was added over ice water; the resulting solid was collected, washed with water, and recrystallized from ethanol and water to produce the intermediate compounds 9a–c. Subsequently, isatin derivatives 9a-c reacted with compound 4 in the same conditions described previously in the preparation of compounds 6a-i.

General procedure for preparation of N-benzylated derivatives of 2-methoxy-6-[(1E)-1–(2-[(3E)-2-oxo-2,3-dihydro-1H-indol-3-ylidene]hydrazin-1-ylidene)ethyl]benzene-1-sulphonamides (12a-c)

Isatin derivatives 5a, 5d and 5f (2 mmol) were refluxed with (2.8 mmol) of benzyl bromide 8 in acetonitrile (20 ml) using a catalytic amount of potassium iodide and dry potassium carbonate (4 mmol). TLC was used in monitoring the reaction progression. After reaction completion, it was added over ice water; the resulting solid was collected, washed with water, and recrystallized from ethanol and water to produce the intermediate compounds 10a-c. Subsequently, isatin derivatives 10a-c reacted with compound 4 in the same conditions described previously in the synthesis of compounds 6a-i.

Biological evaluation

The NCI-USA antitumor assessment³⁹, MTT viability^40,41, VEGFR-2 inhibition^42,43, CA I, II, and IX inhibition studies^44–47, cell cycle analysis and annexin V-FITC apoptosis assay^48,49 were all carried out as previously published in the current study. The supplementary data included descriptions of every experimental technique.

Molecular modeling

Molecular docking studies

The crystal structure of VEGFR-2 (Code: 4ASD)⁵⁰ was retrieved from protein data bank. The docking study was carried out on the co-crystalized ligand (sorafenib) and 12b as a promising inhibitor. Ligands were drawn into Marvin Sketch V19.12⁵¹. The docking was performed using AutoDock Vina⁵² in accordance with our previous report⁵. The active site was defined by the grid box of (x= −23.3, y = 0.1 and z= −10.1) coordinates with size (x = 23.2, y = 18.3, z = 21.6). Using the Discovery studio client, the 3D visualisation and the 2D schematic presentation were produced⁵³.

MD Simulation

The best docking pose was subjected for molecular dynamics (MD) using GROMACS 2021 over 100 ns⁵⁴. The details of applied parameters and procedures were mentioned in the supplementary file.

Disclosure statement

CT Supuran is Editor-in-Chief of the Journal of Enzyme Inhibition and Medicinal Chemistry. He was not involved in the assessment, peer review, or decision-making process of this paper. The authors have no relevant affiliations of financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Large Group Research Project under grant number (RGP2/321/44). The authors extend their appreciation to the Research Center at AlMaarefa University for funding this work.