Synthesis and Biological Evaluation of Oxindole Sulfonamide Derivatives as Bruton's Tyrosine Kinase Inhibitors **

Bruton's tyrosine kinase (BTK) is a promising molecular target for several human B‐cell‐related autoimmune disorders, inflammation, and haematological malignancies. The pathogenic alterations in various cancer tissues depend on mutant BTK for cell proliferation and survival, and BTK is also overexpressed in a range of hematopoietic cells. Due to this, BTK is emerging as a potential drug target to treat various human diseases, and several reversible and irreversible inhibitors have been developed and are being developed. As a result, BTK inhibition, clinically validated as an anticancer treatment, is finding great interest in B‐cell malignancies and solid tumours. This study focuses on the design and synthesis of new oxindole sulfonamide derivatives as promising inhibitors of BTK with negligible off‐target effects. The most cytotoxic compounds with greater basicity were PID‐4 (2.29±0.52 μM), PID‐6 (9.37±2.47 μM), and PID‐19 (2.64±0.88 μM). These compounds caused a selective inhibition of Burkitt's lymphoma RAMOS cells without significant cytotoxicity in non‐BTK cancerous and non‐cancerous cell lines. Further, PID‐4 showed promising activity in inhibiting BTK and downstream signalling cascades. As a potent inhibitor of Burkitt's lymphoma cells, PID‐4 is a promising lead for developing novel chemotherapeutics.


Introduction
Bruton's tyrosine kinase (BTK) is a Tec family kinase that functions as a nonreceptor tyrosine kinase. [1,2]Tec family kinases (TFKs) form the second largest family of cytoplasmic tyrosine kinases in mammalian cells.BTK is critical for B-cell development, differentiation, and signalling. [1,2]Moreover, BTK expression is assumed to be a prerequisite for B-cell proliferation and survival, and BTK-deficient B lymphocytes fail to reach the mature state and undergo premature death. [3]The BTK gene comprises 19 exons and spans approximately 37.5 kb on the human X chromosome. [4]ith an estimated 19.3 million new diagnoses and 10 million deaths worldwide, cancer is one of the leading causes of human deaths as of 2020. [5]The current anticancer drugs each have a different mode of action, ultimately leading to variations in how they affect different cancer types and healthy cells. [6]A significant and practical worry with cancer is the development of resistance to current multi-drug chemotherapy and its poor pace of cure, severe side effects, and the overarching adverse impact on the lives of both patients and caregivers. [6,7]Therefore, developing better and more effective chemotherapeutic drugs with minimal off-target effects is crucial for treating cancer. [8]ngiogenesis, evasion of the anti-tumour immune system response, changes in cellular proliferation, survivability, motility, metabolism, and abnormal protein kinase activity are all critical characteristics of malignancies. [9][12][13][14] BTK was previously found to be mutated in Xlinked agammaglobulinemia (XLA) and also found to be crucial for the development of B lymphocyte development. [15,16]XLA is an immunodeficiency disorder that is inherited and was previously first described by Ogdon Bruton in 1952 and later reported through various research on recurrent bacterial infections.Due to a severe obstruction to B-cell growth in the bone marrow, the patients with XLA have low B cells circulating in their bodies, and the antibody count is nearly negligible in their serum. [17]TK has drawn much attention as a molecular target in medicinal chemistry since preclinical and clinical investigations using small-molecule inhibitors have demonstrated outstanding anti-tumour effects. [18]BTK inhibitors (BTKIs) may be divided into two categories depending on how they bind to BTK, their scaffold designs, and their mechanisms of action.The first category of irreversible inhibitors forms a covalent link with the ATP-binding site of BTK that has the amino acid residue Cys481.21] In 2013, a significant milestone was achieved when ibrutinib, the first covalent and irreversible BTKI, received approval from the US Food and Drug Administration for treating relapsed and refractory MCL. [22]Building on this success, the subsequent years witnessed the development and approval of second-and third-generation irreversible BTKIs, such as acalabrutinib, zanubrutinib, tirabrutinib, and orelabrutinib.These newer agents aimed to reduce the adverse effects ofiIbrutinib while broadening their application to various B-cell malignancies. [22]Simultaneously, BTKIs have seen a surge in research and development, with several irreversible BTKIs currently in various phases of preclinical and clinical trials. [18]hile all five currently approved BTKIs are irreversible in their mode of action, there is a growing interest in exploring reversible inhibitors for treating haematological malignancies and autoimmune diseases. [18]This ongoing exploration of different inhibition mechanisms underscores the dynamic nature of BTK research and its potential for therapeutic innovation.Against this backdrop, the quest for novel BTKIs continues to be an exciting and evolving area within medicinal chemistry.In this context, we report the design, synthesis, and biological evaluation of a series of novel oxindole sulfonamide derivatives as promising anti-BTK candidates.

Chemistry
Within drug discovery, there is a growing interest in harnessing the potential of hybridized bioactive molecules to create novel compounds that surpass the effectiveness of existing options.This approach promises to enhance therapeutic outcomes and mitigate adverse effects associated with current treatments (Figure 1).Among the key players in this endeavour, oxindole sulfonamide has emerged as a pivotal building block, providing a versatile scaffold for developing hybrid candidates with superior efficacy and improved safety profiles.Our research represents a multifaceted exploration into the fusion of bioactive molecules, with a particular focus on oxindole sulfonamide.By coupling this scaffold with Tetrahydro Pyran, we sought to design hybrid compounds with enhanced anticancer properties against BTK-positive cancers.We subjected the resultant hybrid compounds for in vitro biological evaluation and molecular docking studies to elucidate their binding affinities.

Docking studies
A docking study was conducted to gain insights into oxindole sulfonamide derivatives' binding affinity and intermolecular interactions with BTK.BTK complexed with an inhibitor ibrutinib (PDB ID-5P9J), was used for docking analysis.Ibrutinib was used as a standard drug to screen oxindole sulfonamide derivatives.Crystallographic data shows that ibrutinib and zanubrutinib share similar bioactive conformations within the wild-type BTK binding site. [23,24]From these complexes, it was observed that besides the covalent bond with Cys481, the two complexes were mainly stabilized by similar interactions.This includes the cation-π contact between the phenoxyphenyl ring and Lys430 side chain and the hydrogen bonds with Met477 and Glu475 backbones.Noncovalent inhibitors that do not interact with Cys481 can inhibit C481R, T474I and T474 M mutants and represent an attractive therapeutic option. [25]oncovalent BTKIs provide several advantages over existing covalent inhibitors.Though covalent inhibitors lose potency against Cys481 mutants, some noncovalent inhibitors retain potent inhibition against C481S and C481R BTK mutants.This provides a probably practical treatment choice for ibrutinibresistant or naïve patients. [25]e docked all 19 oxindole sulfonamide derivatives into the binding site of BTK.The top 19 compounds were selected based on binding energy score.The molecular docking results include docking binding energy, the interacting amino acid residues in the binding pocket of the protein, and the type of interaction and structure of ligands (Table 2).Binding energies and molecular interactions of oxindole sulfonamide derivatives were compared with those of ibrutinib to select molecules for further studies.Ibrutinib exhibited the highest binding energy to the active site residues of BTK À 10.8 kcal/mol and formed hydrogen bonds with Met 377, Glu375, Pi-cation stacking with Lys 430, Pianion stacking with Asp 539 and PiÀ Alkyl Bond with Val416, which are found essential for interactions in the crystallographic complex.Compared to ibrutinib, all derivatives exhibited binding energy ranging from À 7.9 to À 10.8 kcal/mol.PID-6 exhibited similar binding energy (dock score À 10.8 kcal/mol) to the active site residues of BTK as ibrutinib.The docking scores of PID-4 and PID-19, two other derivatives with better cytotoxicity in BTK-positive cells (see results in subsequent sections), were À 10.3 kcal/mol and 10.7 kcal/mol, respectively.Molecular docking interactions of PID-4, PID-6 and PID-19 are shown in Figure 2. We selected the top 19 compounds with binding energy ranging from À 7.9 to À 10.8 kcal/mol, and showing interactions essential for inhibiting BTK were selected for further cytotoxicity profiling.

In vitro cytotoxic activity
We chose a panel of human cell lines comprising ITK/BTK-null, ITK-positive and BTK-positive malignant cells and ITK/BTK-null

Entry
Amines (3a-3 s) 4a-4 s and PID 1-19 derivatives non-malignant fibroblasts to test the selective cytotoxicity of derivatives.RAMOS and K562 cell lines were positive for BTK and showed no ITK expression; however, BTK expression was relatively higher in RAMOS than in K562 cells (Figure 3A).As for ITK, its expression was higher in JURKAT than in CCRFÀ CEM cells, and both the cell lines were negative for BTK (Figure 3A).Due to high BTK expression levels, RAMOS cells are wellrecognized as a highly positive cell line for BTK. [26,27]Other cell lines from the panel, A549, HCT116, U2OS, MRC-5 and BJ cells, did not express BTK or ITK (Figure 3A).
We next screened all 19 derivatives and ibrutinib, as a positive control, in the cell line panel following a standard procedure at our high throughput screening facility.As expected, ibrutinib showed high cytotoxicity against JURKAT, CEMÀ CCRF and RAMOS cells (Table 3).In addition, it was also toxic to other cancer and non-cancer cell lines.In contrast, our derivatives were inactive in ITK/BTK-null and ITK-positive cell lines, except for PID-4, which had a weak cytotoxic activity in CCRFÀ CEM cells (IC 50 40.87� 7.95 μM) (Table 3).Out of the 19 tested compounds, three showed high (PID-4, IC 50 2.29 � 0.52 μM; PID-6, IC 50 = 9.37 � 2.47 μM; PID-19, IC 50 = 2.64 � 0.88 μM) to moderate (PID-13, IC 50 34.45� 0.37 μM and PID-16, IC 50 30.83� 3.96 μM) activity in BTK-high RAMOS cells.Interestingly, these compounds showed no cytotoxicity in K562 cells, expressing low levels of BTK (Figure 3A), most probably because the bcrÀ abl fusion gene is a known primary driver in K562 cells. [28]Unlike ibrutinib, none of the compounds active in BTKhigh RAMOS cells affected the growth of non-malignant cell lines.Only PID-18, inactive in all cancer cell lines, had moderate toxicity against MRC-5 cells (Table 3).These data show selective cytotoxicity of PID-4, PID-6 and PID-19 against BTK-high cancer

PID-14
cells with no off-target effects in ITK/BTK-null and ITK-positive cell lines.

PID-4 effect on BTK signalling
Inhibition of BTK is a pivotal strategy in the field of molecular medicine, with compounds like ibrutinib and selective kinase inhibitor CHMFLÀ BTK-11 demonstrating their effectiveness in blocking the activation of downstream MAPK family proteins, such as p38, ERK1/2 and JNK. [31,33]This inhibition is particularly significant in THP-1 differentiated macrophages and RAMOS cells, where BTK is critical in orchestrating cellular responses. [26,29]ilding upon this knowledge, we assessed the effect of the three most potent cytotoxic compounds -PID-4, PID-6, and PID-19 -on the inhibition of BTK and the ensuing downstream activation of ERK1/2, JNK, and p38 in RAMOS cells.Among these compounds, our investigation unveiled that PID-4 and PID-19 had a profound effect, significantly inhibiting BTK Tyr223 phosphorylation at a concentration of 50 μM after 24-hour treatment (Figure 3B, C).The inhibition of BTK by the two compounds reverberated further downstream, as evinced by the outcomes of western blot analysis, which showcased a substantial reduction in ERK1/2, JNK, and p38 phosphorylation (Figure 3DÀ G); however, a statistically significant effect was noted only for PID-4 against p38 phosphorylation (Figure 3G).While PID-6 is part of the same series of compounds, it did not

PID-19
affect BTK Tyr223 phosphorylation.This disparity may be attributed in part to PID-6 being three times less cytotoxic to RAMOS cells when compared to PID-4 and PID-19.Interestingly, all three compounds also did not affect ITK levels in JURKAT (Figure 3H), indicating their activity against B-cell receptor signalling.
Since PID-4 showed a significant effect in the above studies, we selected this compound for further biological evaluation.We subjected RAMOS cells to an initial stimulation with lipopolysaccharide (LPS), a known inducer of BTK phosphorylation and the subsequent activation of downstream MAPK family proteins. [29]As expected, LPS stimulation significantly increased activation, as indicated by the increase in BTK, ERK, JNK and p28 phosphorylation (Figure 4A, B).PID-4 treatment of stimulated cells resulted in significant downregulation of BTK Tyr223 phosphorylation (Figure 4A, B).This reduction in BTK phosphorylation was mirrored by a corresponding decrease in the phosphorylation of ERK1/2, JNK and p38 at 10 or 50 μM concentrations.Overall, the findings show a selective effect of PID-4 on BTK and downstream signalling cascades.

Conclusions
We embarked on the synthesis of a novel series of compounds, specifically 3-(dihydro-2H-pyran-4(3H)-ylidene)-2-oxoindoline-5sulfonamide analogues (designated as PID 1-19).This synthesis was achieved through Knoevenagel condensation, wherein dihydro-2H-pyran-4(3H)-one was combined with a range of corresponding sulfonamides (denoted as 4 a-s).Importantly, this synthesis methodology proved highly efficient, consistently yielding these compounds in good quantities.The primary objective of our investigation was to explore the potential of the newly synthesized oxindole sulfonamide derivatives as promising inhibitors of cancer cell lines characterized by overactive BTK.We placed particular emphasis on assessing the selective cytotoxicity of these compounds.Remarkably, our findings indicated that PID-4, PID-6, and PID-19 exhibited high selectivity against BTK-high RAMOS cells, with minimal to no cytotoxicity in ITK/BTK-null cancer cell lines, ITK-positive cancer cell lines, and non-malignant cells.This crucial aspect underscores the specificity of these compounds towards BTK-high cancer cells while sparing non-malignant cells, which is a pivotal feature for potential therapeutic applications.Both PID-4 and PID-19 significantly inhibited pBTK levels in RAMOS cells, with PID-4 standing out as a potent inhibitor of BTK-high cancer cells, demonstrating a proclivity for inhibiting pBTK activities in LPS-stimulated RAMOS cells.This selective action on BTK and its associated pathways positions PID-4 as a promising lead for further exploration and development as a potential drug for malignancies characterized by high BTK expression.This research contributes significantly to the ongoing efforts in the research and development of BTKIs, where efficacy and safety are paramount.

Materials and methods
All reagents and solvents used to synthesize derivatives were purchased from commercial sources (Sigma Aldrich, TCI, combiblocks).All reactions were monitored by TLC using Merck classic aluminium silica plates with size 20×20 cm, thickness 200 μm were detected in UV 254 nm, ninhydrine and poly molbdic acid (PMA) solutions.All compounds were purified in combi-flash chromatography using RediSep RF 1.5 Flash silica gel columns manufactured by Teledyne ISCO.Proton 1 H and 13 C NMR spectra were recorded on a Bruker Avance 300 MHz and Ascend 400 MHz spectrometer.Proton NMR chemical shifts are reported in parts per million (δ) using TMS as a standard reference.HRMS ESI mode positive ion trap detector.IR spectra were recorded on an FT-IR spectrometer (Shimadzu FT-IR 8300 spectrophotometer), and only major peaks were reported in cm À 1 . 1 H NMR, 13 C NMR, and FT-IR spectra of all compounds are provided as Supporting Information.

Procedure for synthesis of 2-oxoindoline-5-sulfonyl chloride (2):
Indolin-2-one (2) (3.00 g, 22.5 mmol) was charged with chlorosulfonic acid (3.0 mL, 45 mmol) at 0 °C dropwise over 10 minutes.After completion addition, the reaction mixture was warmed to room temperature and stirred at 70 °C for 1 h.The reaction mixture was cooled to room temperature and poured into crushed ice (30 g), at which point, solids were precipitated.The obtained solids were filtered and dried under vacuum to obtain 2-oxoindoline-5-sulfonyl chloride (2) (3.75 g, 72 % yield) as a light brown solid: 1  General procedure for synthesis of compound 4 a-s: A solution of 2 (1.0 equiv.) in anhydrous 1, 4-dioxane was added 3 a-s (1.2 equiv.)and pyridine (3.0 equiv.)under Ar atmosphere at room temperature.After complete addition, the reaction mixture was stirred at room temperature for 2 h.The reaction mixture was diluted with water (20 mL), acidified with 2 N HCl solution to pH 6 and extracted with EtOAc (3×30 mL).The combined organic layer was dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure to obtain the crude product.The product was washed with MTBE (20 mL) and dried under a vacuum to obtain compounds 4 a-s.These compounds were directly taken to the next step without purification.

General procedure for synthesis of compounds PID 1-19:
The solution of 4 a-s (1.0 equivalent in EtOH (5.0 mL) was added 5 (1.2 equiv.)and pyrrolidine (3.0 equiv.)under Ar atmosphere at room temperature.After complete addition, the resultant reaction mixture was stirred at room temperature for 3 h.The reaction mixture was diluted with water, acidified with 2 N HCl to pH 4 and extracted with EtOAc (3×50 mL).The combined organic layer was washed with brine solution (20 mL), dried over anhydrous Na 2 SO 4, filtered and concentrated under reduced pressure to obtain the crude product.The product was purified by flash column chromatography eluted with 1-10 % CH 3 OH in dichloromethane to obtain PID 1-19 derivatives with 65 % to 78 % yields.

Preparation of protein/receptor
The crystalline structure of BTK complexed with an inhibitor Ibrutinib (PDB ID-5P9J) with resolution 2.5 Å was downloaded from Research Collaboratory for Structural Bioinformatics (RCSB) which is the protein data bank database (www.rcsb.org), in .pdbformat.The protein was created using the AutoDock programme. [30]It has a single chain, which was chosen as the target of the study.The natural ligand, non-interacting ions, and water molecules were all removed from the crystal structure.In order to reduce the strain on the crystal structure and make the protein usable in the AutoDock docking simulation tool, the lacking hydrogens were added.The protein was created using the UCSF Chimera graphical user interface after the structural reduction, which includes hydrogen atoms, Gasteiger charge calculations, and the merging of the nonpolar hydrogens to carbon atoms.

ChemSketch
ChemSketch is a programme specialized for sketching chemical structures by focusing on chemical structure data.It aids in the two-dimensional drawing of compounds, which can then be quickly transformed into three dimensions using a three-dimensional algorithm that also considers the molecular dynamics of such compounds.In the domains of bioinformatics and cheminformatics, it is extensively employed.ChemSketch has been utilized in order to create analogues of the chemicals.Using UCSF Chimera, ligand input files were created for docking and saved in mol2 file format. [31]

Chimera
The interactions were visualized and examined using UCSF Chimera, which can be extended to analyze molecular structures and associated data such as density maps, sequence alignment outcomes, docking outcomes, and trajectory findings.It offers a high degree of functionality in addition to fundamental functions, such as visualization and extension. [32]

Discovery Studio
The Discovery Studio assists in determining the types of interactions and bond lengths between the active sites in the target and ligand conformations.BIOVIA Discovery Studio Visualizer (Accelrys) is a unified, user-friendly graphical interface for effective drug design and protein modelling.

Docking
The molecular docking method was applied to the selected ligands with the help of AutoDock Vina. [33]A grid box for BTK with the dimensions X:30, Y:30, Z:30 Å and a grid spacing of 1.0 Å focused on X: 18.309, Y:9.2081, Z: 7.9357 was identified as the protein target docking site, and best molecular interacting compounds were observed.The interactions between the active sites in the target and ligand conformation, along with the type of interaction and bond distances, were identified using Discovery Studio Visualizer.

Cell lines
Cells were either purchased from ATCC (Middlesex, UK) or DSMZ (Braunschweig, Germany) and maintained at 37 °C in a humidified incubator (5 % CO 2 /atmospheric air) in recommended growth medium supplemented with 10 % fetal calf serum, antibiotics (100 mg/mL streptomycin and 100 U/mL penicillin), 2 mM gluta-mine, 1 mM NaHCO 3 , 1 mM C 3 H 3 NaO 3 , and 20 mM HEPES.Cell lines were regularly validated and checked for mycoplasma infection every two weeks.

Cytotoxicity assay
The cytotoxicity of compounds was tested using an MTS assay protocol developed for routine compound screening at our facility, and IC 50 values were calculated, as described previously. [33]ug treatment and western blotting analysis RAMOS and JURKAT cells were plated at 0.5×10 6 /mL density in 6well cell culture plates in complete growth media and treated with PID-4, PID-6 and PID-19 at 1-50 μM concentrations for 24 hours.For LPS stimulation, RAMOS cells in a growth medium containing 1 % FCS were exposed to 1 μg/ml LPS for 10 minutes.Cells were next centrifuged to remove LPS-containing media and washed twice with complete growth media containing 10 % FCS.Stimulated cells were next plated at a density of 0.5×10 6 /mL in 6-well plates, followed by treatment with PID-4 for 3 hours.
To prepare whole protein extracts, cells following treatment were collected by centrifugation and lysed in RIPA buffer (Thermo Fisher Scientific, Massachusetts, USA) supplemented with protease (Cat.#04693116001) and phosphatase (Cat.#04906837001) inhibitors from Roche, Basel, Switzerland by sonication on a Cup Horn sonicator (Qsonica, LLC., Connecticut, USA).Sonicated samples were centrifuged at 12 000 RPM for 30 min at 4 °C, and the supernatants were collected in fresh Eppendorf tubes.Thirty-five μg of protein lysates were electrophoresed by 10 % SDS-PAGE and processed for Western blotting following a standard protocol.

Figure 3 .Table 3 .SD
Figure 3.Effect of compounds on BTK signalling.(A) BTK and ITK protein levels in cell line panel.(B) RAMOS cells were treated with PID-4, PID-6 and PID-19 for 24 h, and whole protein extracts were probed for phosphorylated and total BTK by Western blotting.(C) Quantification of pBTK/BTK band intensities.Mean � SEM, n = 2-4, One-way ANOVA, Dunnett's multiple comparison test.(D) Representative blots showing the effects of BTK inhibition on the activation of downstream signalling pathways.n = 2-4.(EÀ G) Quantification of pERK/ERK, pJNK/JNK and p-p38/p38 band intensities.Mean � SEM, n = 2-4, One-way ANOVA, Dunnett's multiple comparison test.(H) JURKAT cells were treated with compounds for 24 hours, and whole protein extracts were probed for ITK expression changes by Western blotting.GAPDH-normalised ITK band intensities are shown below.In B, D and H, the first lane shows compound-untreated cells treated with the same concentration of DMSO (0.5 %) present in the highest tested drug concentration (50 μM).Images of uncropped blots are shown in Supporting Information.

Table 1 .
List of substituents from Scheme 1.

Table 2 .
Molecular docking analysis results using AutoDock Vina for all oxindole sulfonamide derivatives.