5‐Fluoro/(trifluoromethoxy)‐2‐indolinone derivatives with anti‐interleukin‐1 activity

The pro‐inflammatory cytokine interleukin‐1 (IL‐1) drives the pathogenesis of several inflammatory diseases. Recent studies have revealed that 2‐indolinones can modulate cytokine responses. Therefore, we screened several 2‐indolinone derivatives in preliminary studies to develop agents with anti‐IL‐1 activity. First, the putative efficacies and binding interactions of 2‐indolinones were evaluated by docking studies. Second, previously synthesized 5‐fluoro/(trifluoromethoxy)−1H‐indole‐2,3‐dione 3‐(4‐phenylthiosemicarbazones) (compounds 47–69) which had the highest inhibitory effect in the screening were evaluated for inhibitory effects on the IL‐1 receptor (IL‐1R). Compounds 52 (IC50 = 0.09 µM) and 65 (IC50 = 0.07 µM) were selected as lead compounds for the subsequent synthesis of new derivatives. The novel 5‐fluoro/(trifluoromethoxy)−1H‐indole‐2,3‐dione 3‐(4‐phenylthiosemicarbazones) (compounds 70–116) were designed, synthesized, and in vitro studies were completed. The compounds 76, 78, 81, 91, 100, 105, and 107 tested showed nontoxic inhibitory effects on IL‐1R‐dependent responses in the range of 0.01–0.06 µM and stronger than the lead compounds 52 and 65. In vitro and in silico findings showed that compounds 78 (IC50 = 0.01 µM) and 81 (IC50 = 0.02 µM) had the strongest IL‐1R inhibitory effects and the most favorable drug‐like properties. Molecular modeling studies of the compounds 78 and 81 were carried out to determine the possible binding interactions at the active site of the IL‐1R.

or IL-1R3 to form a complex for initiating the response. [8]Once activated, IL-1R1 sends the message downstream, resulting in hyperthermic and inflammatory effects mediated by the release of PGE2 and TNF-α.Since IL-1R1 activation is required to induce and maintain several different immune system-mediated responses, its activity is tightly regulated by redundant mechanisms.Namely, an endogenous receptor antagonist, IL-1Ra, competes with IL-1 for its binding site, thereby suppressing the receptor activation.Additionally, a decoy receptor, named IL-1R2, binds IL-1, but its TIR domain does not activate the acute immune response due to missing catalytic properties.These regulatory mechanisms provide a fine-tuning of the responses to IL-1 by changing the composition of the IL-1 response elements. [9]inically, there are several different approaches used to block IL-1-mediated activity.IL-1R1 activation can be inhibited with synthetic IL-1Ra (anakinra), strengthening endogenous suppressive activity.Secreted IL-1 can also be blocked by either neutralizing antibodies (canakinumab) or recombinant decoy receptor (rilonacept) thus, weakening the IL-1 effects.[12] However, only isunakinra, an IL-1β/IL-1Ra chimeric peptide, has progressed into clinical trials. [13]Small molecules offer an important cost advantage over biological drugs, and they can be more suitable for patients owing to more versatile pharmaceutical forms with better compliance.Thus, developing a safe and effective molecule capable of controlling the effects of IL-1 is of prime importance. [14,15]dole, first isolated in 1866, is a significant ring system found in the structure of various natural products, and on which many recent studies have been carried out due to its pharmaceutical and pharmacological importance.It is the precursor of many active pharmaceutical ingredients such as cyclooxygenase (COX) inhibitors, indomethacin, acemethazine, and etodolac.Tenidap (Pfizer), which carries a 2-indolinone ring is a cytokine modulator and COX/ 5-lipoxygenase (5-LOX) inhibitor [16] (Figure 1).Tenidap and other 3-substituted-2-oxindole-1-carboxamides were first synthesized in 1985 and patented as both COX and 5-LOX enzyme inhibitors. [17]bsequent studies have shown that in addition to COX and 5-LOX inhibitions, it also inhibits the lipopolysaccharide (LPS)-induced synthesis of IL-1 and IL-6 in macrophages. [18,19]Another indole derivative MK-886 (Sandoz) is a selective inhibitor of 5-lipoxygenaseactivating protein (FLAP) that potently suppresses leukotriene biosynthesis (Figure 1). [20,21]In a study examining the effects of some 5-LOX inhibitors on the modulations of IL-1, IL-6, and IL-8 cytokines, it was found that MK-886 is a strong IL-1 synthesis inhibitor.It was determined that the other two cytokines inhibited synovial production to a lesser extent. [22]sed on these findings, research focused on the replacement of the thiophene ring has been carried out to develop new antiinflammatory agents with the 3-substituted 2-indolinone structure.
In this study, IL-1R binding interactions in silico and anti-IL-1 effects in vitro of 5-fluoro/(trifluoromethoxy)−1H-indole-2,3-dione 3-(4-phenylthiosemicarbazones) were evaluated and the leader compounds were determined.The binding interactions and physicochemical properties to predict drug-likeness of the novel compounds designed according to in silico and preliminary in vitro test results were investigated.Based on the in vitro test results, the contribution of substituents at positions 1 and 5 of the indole ring and at positions 3 or 4 of the 4-phenylthiosemicarbazone moiety were determined.
Initially, the energy of the system was minimized for 8000 steps and the system was gradually heated up to 310 K.The annealing step was followed by an equilibration period at constant temperature of 310 K and constant pressure of 1 bar for 10,000 steps.Finally, the molecular dynamics of the equilibrated system was performed for 5000 steps.
Throughout the simulations, a small number of water molecules remained hydrogen bonded to 65. Stationary water molecules were shown in the figure along with the hydrogen bonds.The binding region of compound 65 was similar to the binding sites of IL-1β and anakinra (Figure 3a).It consisted of the following amino acids Phe111, Lys112, Gln113, Val124, Cys125, Pro126, Tyr127, Met128, Glu129, Leu138, and Asp162.This finding underlined the importance of Lys112 for the binding of anakinra to IL-1R (Figures 3b,c and 4). [37,40]Figure 3c showed the best pose of the molecule in interaction with the residues of the protein (these residues were given in Figure 4).

| Analysis of docking results
When we examined all docking poses, it was found that the poses Similarly, compound 81 also displayed hydrogen bonds between N 4 hydrogen and Gln108, and between the lactam carbonyl oxygen of the indole ring with Lys112 (Figure 5c,d).

| Prediction of physicochemical properties
Compliance with Lipinski's rule of five (RO5) [41] and predicted pharmacokinetic properties of the designed and synthesized compounds were determined in silico and compared with the lead compounds 52 and 65 (Table 2).All compounds have molecular weights of less than 500 Daltons, and meet the molecular weight criteria of RO5.According to the criteria of RO5, the numbers of hydrogen bond donors should be less than 5 and the numbers of hydrogen bond acceptors should be less than 10.The number of hydrogen bond donors and acceptors were found to be 1 or 2 and 5-6.with an RO5 value of 0 are the compounds with the best parameters in terms of drug candidate properties.][44][45] The compounds 47-82 and 99 have been previously described by Güzel et al., [30] Karalı et al., [34,46] Sevinçli et al., [39,42] Soylu-Eter et al., [43] Ahmad et al., [44] and Pervez et al. [45] in literature (Experimental information for these molecules was given in the Supplementary Material).Structures of the synthesized new QPlogBB: Blood-brain barrier partition coefficient, CNS: central nervous system activity.QPlogKp: Skin permeability QPlogKhsa: Human serum albumin binding ratio.

| Chemistry
compounds 83-116 were confirmed by spectral (IR, 1 H NMR, 13 C NMR, HSQC-2D, HMBC-2D, and MS) and analytical data (Scheme 1, Section 4, and Supporting Information).The isomeric structures of compounds 72 and 99 were determined by X-ray single-crystal diffraction analysis, and the stable isomers were found to be in Z configuration. [42,43]e new compounds' solid phase (KBr) IR spectra showed in 3134-3361 cm −1 region resulting from the NH stretching bands of the thioamide groups.Furthermore, C═S stretching bands observing 1138-1273 cm −1 region proved the presence of thioamide groups.The mass spectra of new compounds were obtained using the positive electrospray ionization (ESI+) technique, [M+H] + peaks were observed as the base peak in all compounds, and confirmed the molecular weights of the compounds.In addition, [M+H] + +2 peaks were also detected in the spectra of R 3 chlorine or bromine substituted compounds.

| In vitro IL-1 receptor inhibitory effects
We tested the in vitro IL-1R inhibitory effects of the both previously Furthermore, the IL-1-specific SEAP measurements confirmed a potent anti-IL-1 activity of some compounds without affecting the viability of the cells (Tables 3 and 4).  a Defined in the literature. [30,34,39,44,45]orrelated for nonsubstituted derivatives at the R 2 position, which  a Defined in the literature. [39,42,43]ubstitued-compound 52 bearing a chlorine group at position 3 of phenyl ring and 5-(trifluoromethyl) substituted-compund 65 carrying a methyl group at position 4 of the phenyl ring had an IC 50 value at nanomolar range, an expected blocker for IL-1 receptors in "in vitro conditions."Based on the test results, the compounds 52 (IC 50 = 0.09 µM) and 65 (IC 50 = 0.07 µM) with the strongest inhibitory activity and suitable drug properties were selected as the lead compound.However, the effects were significantly reduced in all 1-nonsubstituted compounds 47-50 and 53-63 (Table 3).4).
Both COX-1 and COX-2 contribute to IL-1 beta-stimulated prostanoid synthesis. [47]Although our in vitro screening strategy was based on a specific assessment of the activation of IL-1 receptors through a reporter assay without any interference with COX isozymes, it is worthwhile to evaluate whether new molecules interfere with the COX enzyme.Using an inhibitory screening immunoassay kit, we tested the potential COX-1 inhibitory actions of two compounds with the most favorable IC 50 values, compounds 78 and 81.The COX-1/2 inhibitor indomethacin was used as a positive control.While the known inhibitor, indomethacin displayed an IC 50 value of 0.12 pmol/mL, similar to earlier enzymatic studies, [48] both compounds failed to block COX-1, and IC 50 values cannot be elucidated.Considering the high concentration (30 µM) of the tested compounds, without any success, we decided not to evaluate COX-2 actions at this stage.

| CONCLUSION
In this study, we developed a series of new 5-fluoro/(trifluoromethoxy)−1 inhbitory effects and the most favorable drug-like properties, and can be selected as target compounds for mechanistic and in vivo studies.
Whereas, considering the similarities between TLR2 and IL-1 receptors, and the fact that both IL-1 and TLR ligands produce indistinguishable responses during some important inflammatory events such as the induction of COX or nitric oxide, [49] further studies are needed to discriminate the specificity of the new compounds regarding these two receptor types.

| Molecular modeling
All synthesized compounds were subjected to docking studies on Maestro implemented in the Schrödinger-Small-Molecule Drug Discovery Suite package program (v.2018-3). [50].1.

| Protein selection and preparation
The crystallographic structure of IL-1R1 was obtained from Protein Data Bank (PDB database, www.rcsb.org)with the code 1ITB.pdb(2.5 Å) to determine the binding mode interactions for ligands.This crystal structure includes IL-1R1 in complex with the ligand IL-1β. [40]The crystal structure of the complex was cleaned, native ligand IL-1β and the crystallization water molecules, salts, and other small molecules were removed in order not to affect the protein-ligand binding relations, and all hydrogen atoms were added.The structures were preprocessed, filled in missing loops and side chains, optimized of hydrogenbonded structures, and minimized at pH 7.0 by Protein Preparation Wizard application in Maestro.The IL-1R1 structure was acquired as mae file.

| Ligand preparation
The 2D structures of compounds were drawn by 2D Sketcher application, all hydrogen atoms were added, energy minimization was done at pH 7.0 according to OPLS3e method, and 3D ligand structures were created, and saved as mae file with LigPrep application.

| Molecular docking method
The binding site on IL-1R1 was determined by the model using

| Prediction of physicochemical properties
All compounds were applied to Qikprop application to evaluate compliance with Lipinski's RO5 which is an important standard for the druglikeness and some physicochemical properties relating to absorption, distribution, metabolism, and excretion (ADME). [41] 2) (5 mmol) and anhydrous potassium carbonate (K 2 CO 3 ) (7 mmol, 0.97 g) were continuously stirred in 10 mL of dimethylformamide (DMF) at room temperature for 1 h.After the addition of iodomethane/bromoethane/benzyl chloride (15 mmol) and anhydrous potassium iodide (KI) (1 mmol, 0.17 g) as a catalyst to the mixture, the reaction was heated to 50-60°C under reflux with continuous stirring 1-3 h.The reaction mixture was first evaporated to dryness under reduced pressure to obtain crude product.3][54][55][56] 4.2.3 | General procedure for the synthesis of 4-(substituted phenyl)thiosemicarbazides (28-46)   A solution of substituted phenyl isothiocyanates (compounds 9-27) (5 mmol) in 10 mL of ethanol were added dropwise with vigorous stirring to a solution of hydrazine hydrate (5 mmol) in 10 mL of ethanol with iced water cooling.59][60][61]  (2.5 mmol) in 10 mL of absolute ethanol and trace amounts of concentrated sulphuric acid in ethanol (100 mL) was added occasionally as a catalyst to increase efficiency.The reaction mixture was heated to 80-90°C under reflux until the completion of the reaction.

(
IC 50 = 70 and 90 nM, respectively) on IL-1R inhibition displayed very low binding free energy (−33.73 kJ/mol, QK d : 2.07µM and −32.55 kJ/mol, QK d : 3.27 µM, respectively) and have been selected as the lead compounds for subsequent synthesis of new derivatives.

F
of the compounds which had 3/4-methoxy, 4-(trifluoromethoxy), 4-thiomethyl substituted compounds at R 3 substitution were largely I G U R E 3 In the part of a, the crystal structure complexes of interleukin (IL)-1R1 with IL-1β, anakinra, and compound 65 appear as their surface areas stained with colorful, respectively.In the parts of b and c, best binding conformation of compound 65 and zoomed-in view of this conformation are seen.In part of d, the structures of compounds 52 and 65 are seen.alike with the docking poses of the lead compounds 52 and 65;whereas the compounds which had 3/4-(trifluoromethyl), 3-fluorine or chlorine substituted at R 3 were partially alike.This evaluation was also consistent with the in vitro test results.We observed that the compounds with no substitution at R 2 did not display promising activity.However, the compounds with benzyl moiety at R 2 substitution made it challenging to capture ligand-receptor interactions required for activity potentially due to the larger benzyl group compared to methyl or ethyl substitution.Compound 78 formed two hydrogen bonds, the first between N 4 hydrogen and Ile110, and the second between the methoxy oxygen on the phenyl ring with Val16.In addition, hydrophobic interactions with Phe111, Lys112, and Lys114 amino acids of IL-1R1 were detected with the methyl group of the compound (Figure5a,b).
25, respectively.According to these calculations, all compounds complied with hydrogen bond donor and acceptor groups criteria of RO5.The calculated values for the coefficient of distribution (QPlogP) of the R 1 -fluorine and R 2 -methyl or ethyl-substituted compounds (70-79, 81, 70-79, 81, and 82, except 80), and R 1 -trifluoromethoxy, R 2 -hydrogen or methyl, R 3 -halogen, methyl or methoxy-substituted compounds (92, 93, 98, 99, 102, and 106) are in the range of 3.839-4.924and meet the partition coefficient criteria of RO5.When all these values were examined, it was seen that compounds 70-79, 81, 82, 92, 93, 98, 99, 102, and 106 were suitable for all parameters of RO5 and the noncompliance RO5 numbers of the compounds were zero.Both the most potent anti-IL1R inhibitor compounds 76, 78, and 81, as well as the lead compounds 52 and 65 took part in this group.Whereas, in the R 1 -fluorine-substituted derivatives, the QPlogP values are above 5 for all the R 2 -benzylsubstituted compounds (83-91) and the R 2 -ethyl-substituted compound (80) carrying the trifluoromethoxy group at the thiosemicarbazone moiety.The calculated QPlogP values of the compounds were in the range of 5.066-6.365.In the R 1 trifluoromethoxy substituted derivatives, the QPlogP values of all the R 2 benzyl substituted compounds (113-117) and most of the R 2 -ethyl-substituted compounds (105, 107-111) are above 5.In addition, the QPlogP values of the compounds (94-97, 100, and 101) bearing trifluoromethyl, ethyl, t-butyl, thiomethyl, and trifluoromethoxy at the R 3 position, were calculated above 5 in the R 1 -trifluoromethoxy and R 2 -methyl-substituted derivatives.The noncompliance RO5 numbers of the compounds are one.Therefore, all described compounds have sufficient properties to drug-likeness, and there was no problem in agreement with RO5 for all the compounds as a maximum of one rule is allowed not to comply with at this system.The predicted values of solubility in water (QPlogS), blood-brain barrier partition coefficient (QPlogBB), central nervous system (CNS) activity, skin permeability (QPlogKp), ratio of binding serum albumin in human (QPlogKhsa) and oral absorption percentage in humans were also examined.The QPlogS values should be in the range of (−6.5) to 0.5.The QPlogKhsa values were determined in the range of (−5.513) to (−6.477) for the compounds 52, 71, 72, 74, 75, 77-79, 81, 82, 98, and 99, as well as the lead compound 52, and complied with this criterion in terms of water solubility.Since the other compounds and the lead compound 65 have high lipophilicity, QPlogS values were found to be (−6.575) to (−8.951).The CNS activity of the compound whose QPlogBB value is close to positive values increases, so it can easily cross the blood-brain barrier.The QPlogBB value and CNS activity should be in the ranges of (−3) to 1.2 and (−2) to 2, respectively.The QPlogBB values and CNS activities of the compounds 70-116, as well as the lead compounds 52 and 65 are on average within the reference ranges from −0.332 to 0.172 and 0 or 1, respectively.These values indicate that most of the compounds have low CNS activities.The QPlogKp and QPlogKhsa values should be in the ranges of −8 to −1 and −1.5 to 1.5, respectively.The values of the compounds 52, 65, and 70-116 were suitable for reference with the ranges of (−2.358) to (−0.530) and 0.273-0.951,respectively.The oral absorption percentage F I G U R E 4 Interactions established by compound 65 with the interleukin (IL)-1R1 protein. in human has been defined as >80 high and <25 weak and the values were calculated 100% for the compounds 52, 65, and 70-116.These findings show that compounds 71, 72, 74, 75, 77-79, 81, 82, 98, and 99

F I G U R E 5
Binding modes and interactions of compounds 78 (a, b) and 81 (c, d) with interleukin (IL)-1R1 (PDB ID: 1ITB).In the parts of a and c, compound 65 was seen in white, compound 78 in pink, and compound 81 in green to compare.In the parts of b and d, the distances of the interactions were written near the interactions on the figures.T A B L E 2 Lipinski's rule of five and predicted pharmacokinetic properties data of 5-fluoro/(trifluoromethoxy)−1H-indole-2,3-dione 3-(4-substituted phenylthiosemicarbazones) 52, 65, QPlogP: Partition coefficient QPlogS: Solubility in water (S: concentration in saturated solution in water [mol/dm 3 ]).

1 H
NMR spectra displayed the thioamide N 2 and N 4 protons of the thiosemicarbazone moiety as two different singlets at δ 12.55-12.72ppm and 10.80-11.06regions, respectively.While the indole proton signals of the compounds 83-91 bearing the fluorine group at the position 5 of the indole ring were observed at low ppm values due to the strong electron donor effect of fluorine, the indole proton signals of the compounds 92-116 bearing the trifluoromethoxy group at the position 5 of the indole ring were observed in the high ppm region.Resonance values of aromatic protons were determined by considering 1 H-13 C relationships and substituent effects in HSQC-2D and HMBC-2D spectra.In the spectra of the compounds 83-91, indole C 4 -H signals were observed at δ 7.66-7.71ppm region as a doublet of a doublet.The indole C 6 -H signaled at δ 7.20-7.23ppm as a triplet of doublet, while the indole C 7 -H signaled at δ 7.03-7.04ppm as a doublet of a doublet.In the spectra of compounds bearing the trifluoromethoxy group at position 5 of the indole ring, indole C 4 -H signals were observed at δ 7.77-7.88ppm as a doublet except the compound 104 as singlet.The indole C 6 -H was observed as a multiplet at δ 7.36-7.50ppm alone or at δ 7.36-7.43ppm with some phenyl protons or as a doublet of doublet at δ 7.34-7.62ppm or signaled at δ 7.45-7.48ppm as a doublet of doublet of doublet, too.The indole C 7 -H signaled at δ 7.01-7.33ppm as a doublet.The presence of singlet and single peaks of thioamide N 2 and N 4 proton signals, as well as a single signal of the phenyl protons, and singlet and single peaks of the methyl, ethyl, methoxy, and thiomethyl groups in the phenyl ring confirmed that the compounds were eluded only in Z-isomer form.All new compounds were subjected to 13 C NMR (decoupled) taken in DMSO-d 6 medium analysis to verify the 1 H NMR findings of the synthesized compounds.The indole ring and thiosemicarbazide residue carbon signals were determined in the spectra of all compounds, and the confirmation by the interaction constants of the interruption of the indole and phenyl signals by 13 C-19 F S C H E M E 1 Synthesis of 5-fluoro/(trifluoromethoxy)−1H-indole-2,3-dione 3-(4-phenylthiosemicarbazones) (compounds 47-116
The novel compounds 70-116 were designed and synthesized based on preliminary in vitro test results of the compounds 47-69, binding interactions to IL-1R and some drug-like physicochemical properties in silico.All compounds tested displayed nontoxic inhibitory effects at IC 50 values in the range of 10 nM to 13 µM.In vitro test results of compounds 70-116 generally showed that methyl or ethyl substituted compounds at position 1 of the indole ring (R 2 ) were more effective than nonsubstituted and benzyl substituted compounds.Most compounds bearing methyl or ethyl groups at position 1 of the indole ring (R 2 ) demonstrated IC 50 values in the nanomolar range.1-Benzyl substituted compounds had higher inhibitory effects than nonsubstituted compounds, but mostly lower inhibitory effects than methyl and ethyl substituted compounds.In addition, these compounds (except 91) were found to have significantly lower potency compared to the lead compounds 52 and 65.Compounds 76, 78, 81, 91, 100, 105, and 107 showed inhibitory effects on IL-1R-dependent responses in the range of 0.01-0.06µM and stronger than the lead compounds 52 and 65.Generally, these compounds are 1-ethylsubstituted compounds (except 1-benzyl substituted 91 and 1-methyl substituted 100).Test results showed that compounds 78 (IC 50 = 0.01 µM), 81 (IC 50 = 0.02 µM), 100 (IC 50 = 0.02 µM), and 107 (IC 50 = 0.02 µM) had the strongest IL-1R inhbitory effects.Among these compounds, only compound 100 carries a methyl group at the R 2 position, whereas all other compounds 78, 81, and 107 are ethyl substituted derivatives at the R 2 position.The results show that 3-methoxy, 3-fluorine, 4-thiomethyl substitutions at the R 3 position in the compounds are important for strong inhibitory activity.It is also seen that the trifluoromethyl group at the R 3 position also contributes significantly to the efficiency.The IC 50 values of compound 76 bearing fluorine at R 1 and 4-trifluoromethyl at R 3 , and R 3 −3-trifluoromethyl substituted compound 105 bearing trifluoromethoxy at R 1 and 3-trifluoromethyl at R 3 are 0.03 and 0.06 µM, respectively.Compounds with benzyl substituent at R 2 showed high IC 50 values, and docking experiments did not support the benzyl group at R 2 as best fit the receptor except the compound 91 carrying fluorine at the R 1 and 4-bromine at the R 3 .The IC 50 value of this compound (0.06 µM) is one of the lowest among 1-benzyl-5-fluorine substituted compounds.Another important finding obtained in this study is that, generally, the inhibitory effects are significantly reduced by 3-methyl, 3-methoxy, 4-ethyl, and 4-trifluoromethoxy substitutions at R 3 in R 1 -fluorine or trifluoromethoxy-R 2 -ethyl substituted compounds (Table 3-dione 3-(4-phenylthiosemicarbazones) based on the lead compounds 52 and 65 through findings of molecular docking studies and in vitro tests.We evaluated the contribution of substituents at positions 1 and 5 of the indole ring and positions 3 and 4 of the 4-phenylthiosemicarbazone moiety to inhibition of IL-1R.Test results showed no significant difference between fluorine and trifluoromethoxy substituted compounds at position 5 of the indole ring, while methyl or ethyl substituted compounds at position 1 of the indole ring had significantly higher inhibitory efficacy than nonsubstituted and benzyl compounds.In addition, it was determined that most of the compounds containing 3 or 4-trifluoromethyl, 3-methoxy, 4-thiomethyl, 3-fluorine, and 4-bromine groups at the 4-phenylthiosemicarbazone moiety were significant inhibitors of IL-1R.The compounds 76, 78, 81, 91, 100, 105, and 107 had stronger nontoxic inhibitory effects on IL-1R-dependent responses than the lead compounds 52 and 65.In vitro and in silico findings showed that compounds 78 and 81 had the strongest IL-1R

the most effective lead molecule 1 -
methyl-5-(trifluoromethoxy) −1H-indole-2,3-dione 3-[4-(4-methylphenyl)-thiosemicarbazone] (compound 65) as a template according to the results of in vitro tests with novel 2-indolinone derivatives correlated with the free binding energy levels to IL-1R1 in in silico docking experiments.This region was similar to the regions of which IL-1β and IL-1 receptor antagonist anakinra bind, and it presented the importance of Lys112 amino acid in binding.The grid was generated in the center of Lys112 amino acid by Receptor Grid Generation application.The prepared ligands were docking on protein structure with XP scanning method in Glide application.Each ligand was screened 50 times to find high-score binding poses, and five poses showing the highest score for each ligand were recorded for analysis.