Discovery of novel enasidenib analogues targeting inhibition of mutant isocitrate dehydrogenase 2 as antileukaemic agents

Abstract Mutant isocitrate dehydrogenase (IDH) 2 “IDH2m” acquires a neo-enzymatic activity reducing α-ketoglutarate to an oncometabolite, D-2-hydroxyglutarate (2-HG). Three s-triazine series were designed and synthesised using enasidenib as a lead compound. In vitro anticancer screening via National Cancer Institute “NCI” revealed that analogues 6a, 6c, 6d, 7g, and 7l were most potent, with mean growth inhibition percentage “GI%” = 66.07, 66.00, 53.70, 35.10, and 81.15, respectively, followed by five-dose screening. Compounds 6c, 6e, and 7c were established as the best IDH2R140Q inhibitors compared to enasidenib, reporting IC50 = 101.70, 67.01, 88.93, and 75.51 nM, respectively. More importantly, 6c, 6e, and 7c displayed poor activity against the wild-type IDH2, IC50 = 2928, 2295, and 3128 nM, respectively, which implementing high selectivity and accordingly safety. Furthermore, 6c was screened for cell cycle arrest, apoptosis induction, and western blot analysis. Finally, computational tools were applied to predict physicochemical properties and binding poses in IDH2R140Q allosteric site.


Introduction
Cancer is considered one of the most threatening diseases devasting the human health.It is expected that by 2040, the number of new cancer cases rise to 29.5 million per year and to 16.4 million cancer-related deaths 1 .Global Cancer Observatory estimated 474 519 new cases of leukaemia worldwide and 5231 in Egypt in the latest estimate 2 .Acute myeloid leukaemia (AML) is a type of leukaemia in which bone marrow produces large number of abnormal blood cells.It is characterised by clonal enlargement of myeloid, which forms all blood cells, with reduced capacity for differentiation.Once it's a build-up in the bone marrow, causes restriction of traditional blood cells production 3 .According to American Cancer Society (ACS), AML is considered the most common cancer type of leukaemia in adult and represents 1 out of 3 cases diagnosed with leukaemia in childhood and teens 4 .The evolution of new efficient therapeutic agents with anticancer properties to overcome this condition is a major objective in medicinal chemistry.It is well established that tumour initiation and maintenance are dependent on metabolic reprogramming of cancer cell [5][6][7] .This means, the tumour cells use altered metabolic pattern compared to normal tissues 5,8 .Isocitrate dehydrogenase (IDH) is a vital metabolic enzyme in the Krebs cycle.It has three isoforms, IDH1 found in cytoplasm, IDH2, and IDH3 located in mitochondria 9 .Normally, IDH2 converts isocitrate by oxidative decarboxylation to alpha-ketoglutarate (a-KG) using NADP þ or NAD þ as cofactors 10 .Point mutations in the active site of IDH2 affecting Arg140 or Arg172 (R140 or R172) result in multiple tumours including; low-grade gliomas, secondary glioblastomas (GBM), angioimmunoblastic T-cell lymphomas, myelodysplastic syndrome (MDS), and AML [11][12][13] .The mutant IDH2 acquires a neo-enzymatic activity that reduces a-KG by NAPH to the oncometabolite called, D-2-hydroxyglutarate (2-HG) 14 .In consequence, accumulation of high levels of 2-HG competitively inhibit dioxygenases like histone and DNA demethylases and proteins that regulate cellular epigenetic status as displayed in Figure 1 10,15 .This epigenetic dysregulation leads to impairment of haematopoietic differentiation 16,17 .
Enasidenib is a first in class and only FDA-approved inhibitor of mutant IDH2 for the treatment of adult patients with relapsed or refractory AML 34 .Enasidenib has lowered 2-HG levels in blood of AML patients, reduced blast counts and increased myeloid cells percentage 24 .Dual tail approach is one of the most effective approaches for compounds design which has been widely applied in some anticancer targets [35][36][37] .As disclosed cocrystal structure of enasidenib with IDH2 R140Q (PDB ID: 5I96), Enasidenib binding is anchored by multiple hydrogen bonds (H-bonds) formed with Q316 and many hydrophobic interactions 24 .It was noticed that binding site of enasidenib is a symmetric and hydrophobic pocket 24 .The symmetric hydrophobic pocket is located within homodimer interface.Herein, for assessment of the dual tail strategy in case of IDH2, we designed and synthesised 28 novel s-triazine inhibitors with symmetric hydrophobic tails using enasidenib as a lead compound.Enasidenib structure is composed of four integrated moieties: s-triazine as a central core, hydroxy alkyl amine as a head, and two lipophilic tails either with or without linkers 24,32 .Relying on these features, we reported three novel series.In series (I), 12 new target compounds, 6a-l, were constructed with a central s-triazine ring bearing morpholine, as bioisostere of hydroxy alkyl amine, and two groups of substituted aromatic rings as the lipophilic symmetric tails connected to the s-triazine core with a methylene hydrazine linker.Regarding series (II), 12 novel compounds, 7a-l, were designed as analogues to series (I) with piperidine as the heterocyclic head as bioisostere of morpholine.In series (III), 4 new target compounds, 8a-d, were assembled with the crucial s-triazine scaffold, morpholine head, and two symmetric lipophilic tails connected to s-triazine ring with diverse manipulated linkers as shown in Figure 3.

Chemistry
Target compounds, of series (I), 6a-l, and series (II), 7a-l were prepared as displayed in Scheme 1.The three chlorine atoms of cyanuric chloride, 1 disclosed diverse reactivity and can be substituted gradually at different temperatures 38 .Compounds 3a,b were prepared from cyanuric chloride 1 via nucleophilic substitution of the first chlorine atom with morpholine, 2a or piperidine, 2b at 0-5 C to afford analogues, 3a and 3b, respectively.Compounds 3a,b underwent nucleophilic substitution of the remaining two chlorine atoms with two hydrazine groups through heating under reflux with excess amount of hydrazine hydrate to provide the trisubstituted s-triazine derivatives, 4a,b, respectively.Condensation of hydrazine derivatives, 4a,b with various aldehydes yielded the corresponding hydrazones, 6a-l and 7a-l, respectively.
Target compounds in series (III), were synthesised as depicted in Scheme 2. Synthesis of 8a and 8b were accomplished through condensation of substituted hydrazine, 4b with two different ketones; p-nitroacetophenone and 5-chloroisatin respectively.Nucleophilic aromatic substitution reaction between compound, 3b and excess amount of either phenylalanine methyl ester or benzoyl hydrazine afforded analogues, 8c and 8d, respectively, bearing two variable linkers.5-Chloroisatin and benzoyl hydrazine were synthesised as previously reported methods 39 .The chemical structures of synthesised compounds were confirmed by elemental analysis, 1 H and 13 C NMR, as well as mass spectrometry.
In this study, twenty-eight compounds were synthesised, whereas their chemical structures were confirmed via 1 H NMR, 13 C NMR, NOESY, elemental analysis as well as EI-MS (Supplementary file contains spectra). 1H NMR spectra of target compounds in series (I) and (II) displayed a characteristic singlet of the imine proton (-N ¼ CH) in the range, 7.96-8.63ppm with concurrent disappearance of hydrazine-NH 2 signal of 4a,b at 4.1 ppm which postulated the formation of imine (-N ¼ CH) bond.Moreover, a characteristic downfield singlet signal at 10.53-13.34ppm corresponding to the (-NHN ¼ C-) proton was observed.Compounds, 6a, 6j, 7a, and 7j were characterised by manifestation of additional downfield signals of (-OH) group at 9.15-11.31ppm, whereas analogues, 6g, 6k, 7g, and 7k displayed singlets of aliphatic (CH 3 /CH 2 ) protons at 3.02, 6.12, 2.97, and 6.08 ppm, respectively.Concerning 1 H NMR spectra of series (III), compound 8a was characterised by upfield singlet at 2.38 ppm related to the aliphatic methyl protons (N ¼ C-CH 3 ).In addition, compound 8b, revealed the (NH) proton of isatine moiety, as a singlet further downfield at 10.88 and 11.37 ppm.Besides, a doublet of NH proton in compound, 8c was recognised at 8.49 ppm.Finally, compound, 8d showed two singlets concerning the (NH-NHCO) and (NH-NHCO) protons assigned at 8.89 and 10.26 ppm, respectively.The geometry of synthesised hydrazones (6a-l and 7a-l), was confirmed as E-isomer rather than Z-isomer relying on interpretation of the 2D NOESY spectra achieved for compound 6i. 1 H-1 H homonuclear NOESY spectrum discovered a NOE signal which is assigned between two protons; H 1 (CH ¼ N, d ¼ 8.18 ppm) and H 2 (NH-N ¼ C, d ¼ 11.39 ppm), which is in agreement with the E-isomer (E-6i) configuration and is not tolerable in the putative (Z-6i) attributable to the larger intramolecular H-H distance of Z-isomer as demonstrated in Figure 4. Therefore, target compounds in series (I) and (II) are established as the E-isomers 40,41 .

Biological evaluation
In vitro anticancer screening at National Cancer Institute (NCI-USA) Target compounds of series (I); 6a-l, (II); 7a-l and (III); 8a-d, were submitted to National Cancer Institute "NCI" (www.dtp.nci.nih.gov).Our target compounds were accepted for assessment of anticancer activity in a single-dose test.Accordingly, five analogues (6a, 6c, 6d, 7g, and 7l) were further subjected to fivedose experiment.In vitro preliminary cytotoxic activity at a single dose of 10 lM against full NCI 60 cancer cells panel Initially, the anticancer effects of target compounds have been assessed at a single (10 lM) dose.The obtained data have been reported as mean-graph of the percent growth (G%) of the treated cancer cells.Percentage growth inhibition (GI%) is calculated (100 À G%) and pronounced in Table 1.Inspection of in vitro antitumor screening data revealed that our s-triazine derivatives exposed variable anticancer activity ranging from low, moderate, to high potency.Preliminary examination of NCI data showed that compounds in series (I) with morpholine head and their corresponding analogues in series (II) with piperidine moiety demonstrated an overall comparable activity as reported in Figure 5. Target compounds in series (III) with manipulated linkers disclosed lower potency.Superiorly, eight s-triazine derivatives from the three series (6a, 6c, 6d, 7a, 7g, 7i, 7l, and 8b) have shown potent broad-spectrum anticancer activity against most the examined cell lines, whereas most of the remaining triazine derivatives, 20 derivatives, have exerted selective anti-proliferative actions towards certain cancer cell lines (Supplementary).The GI% exerted by the examined s-triazine derivatives 6a, 6c, 6d, 7a, 7g, 7i, 7l, and 8b have been listed in Table 1.
Each series pronounced its own structure-activity relationships (SARs) as following: I. Anticancer activity of series (I) was largely affected by substituents on the symmetric lipophilic tails.SAR was deduced as following depending on GI % values demonstrated in Table 1: Scheme 1. Synthesis of target compounds in series (I), 6a-l and series (II), 7a-l.Reagents and conditions: i: Acetone, Na 2 CO 3 , 0-5 C, 4 h; ii: Acetonitrile, hydrazine hydrate 99%, heat under reflux; iii: Ethanol, glacial acetic acid, and heat under reflux.
III.In case of series (III), the corresponding hydrazones of acetophenone, 8a (GI %¼ 23.27), and isatin, 8b (GI% ¼ 47.89) displayed moderate anticancer activity.Isatin tails enhanced the potency compared to acetophenone.Furthermore, introduction of amino acid ester as lipophilic tails in 8c or benzohydrazide in 8d abolished activity as recorded in Table 2. SAR of target compounds of series (I), (II), and (III) is summarised in Figure 6.

In vitro isocitrate dehydrogenase 2 enzyme inhibition assay
The most active cytotoxic analogues, 6a, 6c, 6d, 7g, and 7l as well as other selected compounds, 6e, 6g, 6l, 7a, 7c, 7d, and 7e were evaluated for their abilities to inhibit the IDH2 R140Q mutant via an enzyme-based assay.Enasidenib was used as a positive control and the results were expressed as IC 50 values as presented in Table 3. Compounds 6c, 6e, and 7c established the best enzyme inhibition activity with IC 50 ¼ 101.70, 67.01, and 88.93 nM, Table 1.Preliminary anticancer effects of single dose (10 lM) of s-triazine derivatives 6a, 6c, 6d, 7a, 7 g, 7i, 7l, and 8b against 60 human subpanel cancer cell lines declared as the percentage cell growth inhibition (GI%).respectively, compared to enasidenib which reported IC 50 ¼ 75.5 nM.Accordingly, analogue 6e was the strongest IDH2 R140Q inhibitor and was more active than the standard drug, enasidenib.

Growth inhibition percentage (GI
To evaluate the selectivity profile of our inhibitors, compounds 6c, 6e, 6l, 7a, 7c, 7e, and 7l were selected for enzyme inhibition assay against wild type IDH2.The results confirmed that these inhibitors selectively inhibit IDH2 R140Q by 21-59-fold more than the wild type which gives an early indication on the safety of these inhibitors towards normal cells. In vitro cytotoxicity against human normal cells (human embryonic kidney) Safety profile of compound 6c, as a cytotoxic agent, was investigated towards normal cells, human normal embryonic kidney cells (HEK-293), in comparison to a reference drug, staurosporine 42,43 .

In vitro cell cycle analysis
Targeting cancer cell cycle has been emerged as a viable approach for cancer treatment 44 .To recognise the role of compound 6c in GI of cancer cells, and induction of apoptosis in different phases, DNA flow cytometric analysis was performed to measure the effect of compound 6c on cell cycle progression for leukaemia, HL-

Compounds
Series I Series II  iodide (PI) and then analysed by flow cytometer.The results are reported in Table 5.
A significant increase in the percentage of apoptotic cells at the pre-G1 phase (28.02%) upon exposure to compound 6c by 14fold compared to DMSO (1.95%), was identified with concurrent decrease in the G2/M phase (4.76%) for 6c relative to DMSO (19.79%).Furthermore, an increase in the cells at S and G0-G1 phases (41.18 and 54.06%, respectively) was detected compared to control (33.92 and 46.29%, respectively) as shown in Figure 7. Arresting cell growth in G1/S phase and alteration of the pre-G1 phase are considered significant remarks for compound 6c to induce apoptosis in HL-60(TB) cancer cells.

Detection of apoptosis
Extrinsic as well as intrinsic apoptosis in leukaemia, HL-60(TB) cancer cells, induced by compound 6c was evaluated by Annexin V and PI staining.Herein, HL-60(TB) cells were incubated with compound 6c at GI 50 (158 nM) concentration for 24 h.Compound 6c induced an early apoptosis (2.88%), in HL-60(TB) at 24 h compared to control (0.51%) and enhanced late apoptotic induction (15.75%) by more than 85-fold over the untreated cells (0.18%).Moreover, compound 6c induced necrosis by 7 times more than control as declared in Figures 8 and 9.This determination was consistent with the data obtained from cell cycle analysis revealed in Figure 7.

Western blot analysis for apoptotic markers, Caspase 3 and Caspase 9
Cysteine-containing aspartic acid-specific proteases, caspases provide essential links in cell controlling the apoptotic machinery 45,46 .For this reason, this study was further extended to investigate the mechanism of compound 6c to provoke apoptosis in HL-60(TB) leukaemia cell line.Treatment of HL-60(TB) cancer cells with 6c significantly induced the expression of active caspases 3 and 9 by about 3 and 4-fold, respectively, in comparison to the control (Figure 10).In silico studies   crystal structure of IDH2 R140Q (PDB: 5I96), consists of homodimer bound to NADPH, Ca 2þ and enasidenib.Within the homodimer interface, the allosteric binding site is located 23 .The pocket is encapsulated by four helices (a9, a10, a9', a10') lining the sides, two loops (L1 and L1'), and the Tyr311-Asp312 interaction pairs capping the ends.The binding scores (S) and amino acids in the allosteric site of IDH2 R140Q that interact with inhibitors 6c, 6e, 7c, and enasidenib are illustrated in Table 6.Enasidenib, the co-crystallised drug, is a non-competitive inhibitor 24 .Enasidenib binds by multiple H-bonds and hydrophobic interactions.The 2-methyl-2propanol moiety donates a H-bond to Gln316, while Gln316' accepts a H-bond from linker amine, in addition to forming a Hbond with s-triazine core nitrogen.Additional H-bond between enasidenib's trifluoromethyl moiety and Asp312' is observed as shown in Figure 11(A).Other hydrophobic interactions from surrounding hydrophobic residues include, Trp164', Val294', Val297', Try311, Try311', Asp312', Val315, Val315', Gln316', Ile319', and Leu320'.Molecular docking of 6c verified three H-bond between 6c and IDH2 R140Q where the s-triazine core and one NH group of the linker formed two H-bonds with amide and carbonyl of Gln316' and Gln316, respectively, while H-bond between 6c imine carbon and carbonyl of Gln316' was recognised.The aromatic tails formed van der Waals attraction forces with Leu160, Trp164, Val294, Val297, Leu198, Ile319, and Trp306.Moreover, morpholine head established hydrophobic interaction with Val315 as demonstrated in Figure 11(B).Docking mode of 6e revealed 3H-bonds with Gln316 and Val315 where two H-bonds with NH group and s-triazine nitrogen were observed.The third H-bond was established with NH group of the second linker.While 3-fluorophenyl group of compound 6e formed halogen bond with Ileu319.In addition, compound 6e displayed hydrophobic attraction forces between the aromatic tails and amino acid residues, Leu160', Trp164', Val315', and Ile319'.Another hydrophobic interaction between morpholine carbons and Val297, Leu298, and Ile319, is reported as demonstrated in Figure 11(C).
While compound 7c adapted two more polar interactions than 6c which were one H-bond between Gln316' and NH group while the second was halogen bond between bromine and two surrounding residues, Gln316' and Leu320' as exposed in Figure 11(D).Analogues 6c, 6e, and 7c disclosed parallel layout upon alignment with enasidenib in the allosteric site of IDH2 R140Q as illustrated in Figure 12.

In silico physicochemical, pharmacokinetic prediction, and PAINS filters
To predict the physicochemical and drug-likeness properties of the most potent compounds 6a, 6c, 6d, 7g, and 7l based on NCI in vitro results, SwissADME free online web tool was applied (http://www.swissadme.ch/,accessed on 24 July 2022).The brain or intestinal estimated permeation (BOILED-Egg) model was  developed by calculating both lipophilicity using the Wildman log P method (WLOGP) and polarity expressed in topological polar surface area (TPSA), followed by plotting the relationship between them in a BOILED-Egg diagram 47 .Therefore, we can predict both gastrointestinal absorption and BBB permeability for the tested compounds.The obtained results predicted that physicochemical and pharmacokinetics of the five compounds were in acceptable ranges.All five compounds appeared in the zone of human intestinal absorption (HIA) with no blood-brain barrier (BBB) permeability.Hence, they have high oral bioavailability with a privilege of having a good CNS safety profile.BOILED-Egg was assembled for the tested compounds as illustrated in Figure 13.
In addition, compounds 6c, 6d, 7g, and 7l were not P-glycoprotein substrates (Pgp-); thus, they are not susceptible to the efflux mechanism by the Pgp transporter, which is a mechanism that emerged by some tumour cells as a drug resistance strategy 48 .Compound 6a was predicted to be a P-glycoprotein substrate.Furthermore, SwissADME revealed that compounds 6c, 6d, and 7g fulfilled Lipinski (Pfizer), Veber (GSK), and Egan (Pharmacia) filters predicting that these compounds have promising drug-likeness profiles (Table 7).
Moreover, the bioavailability radar which composed of the calculation of six parameters including size, lipophilicity, polarity, saturation, flexibility, and solubility showed that these compounds (represented by red lines) are almost predicting acceptable oral bioavailability with 7g revealed the best fit among these compounds (Figure 14).
What's more, swissADME-achieved data classified compounds 6c, 6d, and 7l as non-PAINS (pan-assay interference compounds), signifying the high selectivity of our target compounds.

Conclusion
Three novel series of s-triazines were designed and synthesised as inhibitors of IDH2 R140Q relying on the structural futures of the marketed Enasidenib and applying the symmetric dual-tail approach.In series (I), 12 new compounds 6a-l were constructed with a central s-triazine scaffold with morpholine, as the heterocyclic head, and two groups of substituted aromatic rings as the lipophilic symmetric tails connected to the s-triazine core with a methylene hydrazine linker.Regarding series (II), 12 novel compounds, 7a-l, were designed as analogues to series (I) with piperidine as the heterocyclic head.In series (III), 4 new target compounds, 8a-d, were assembled with s-triazine scaffold, morpholine head, and two symmetric lipophilic tails connected to striazine ring with diverse manipulated linkers.Target compounds were assessed as anticancer agents against a panel of sixty cancer cell lines, following the US-NCI protocol.Relying on NCI single-dose screening outcomes, compounds in series (I) and their corresponding analogues in series (II) demonstrated an overall comparable activity, while Target compounds in series (III) disclosed lower potency.The most active analogue, 6c displayed high antiproliferative activity against NCI leukaemia subpanels with GI 50 in nanomolar range and very good activity in inhibiting IDH2 R140Q enzyme (IC 50 ¼ 101.7 nM) while minimal inhibition was reported against wild type (IC 50 ¼ 2928 nM) in vitro.Moreover, 6c showed better safety as a cytotoxic agent towards normal embryonic kidney cells (HEK 293), IC 50  ¼ 53.69 lM in comparison to staurosporine, IC 50  ¼ 35.33 lM.Cell cycle analysis was performed to measure the effect of compound 6c on induction of cell cycle for leukaemia HL-60(TB) cancer cells.It arrested cell growth in G0-G1 and S phases with alteration of the pre-G1 phase.Moreover, compound 6c induced necrosis by 7 times more than the control (DMSO) against HL-60(TB) cancer cells.Treatment of HL-60(TB) cancer cells with compound 6c significantly increased the expression levels of the apoptotic markers, active Caspases 3 and 9 by about 3-and 4-fold, respectively, in comparison to the control.Computational docking study for selected compounds demonstrated strong binding interactions with variable amino acid residues in the allosteric site of IDH2 R140Q .Moreover, pharmacokinetic, and physicochemical properties of the selected inhibitors revealed high predicted oral bioavailability and promising drug-likeness profiles.

General
All organic reagents used were obtained from Alfa Aesar, Sigma-Aldrich, or Merck Company and were used without any further purification.Melting points were determined on an electrothermal melting point apparatus (Stuart SMP10) by the open capillary method and were reported uncorrected.Reactions were monitored by TLC using pre-coated sheet (Fastman Kodak Co., Rochester, NY; Silica 60 F 254 ) using developing systems, chloroform: ethanol and n-hexane: ethyl acetate and were visualised using UV lamp at 254 nm.Elemental analysis was carried out by Perkin-Elmer 2400 CHNS analyser and the results were obtained within ± 0.40 of the theoretical values.Elemental analysis was performed by the regional centre for mycology and biotechnology, Al-Azhar University, Cairo, Egypt. 1 H, 13 C NMR, NOESY spectra were recorded on Bruker FT-NMR spectrometer at (400 MHz) and (100 or 125 MHz), respectively using DMSO-d 6 as a solvent.Values of chemical shift, coupling constants, J, and multiplicity (s were reported in ppm and in Hertz (Hz), respectively. 1H and 13 C NMR experiments were performed by Faculty of Pharmacy and Faculty of Science, Mansoura University, Mansoura, Egypt.Electron ionisation mass spectra (EI-MS) were recorded on Thermo Scientific, ISQ Single Quadruple MS, with ionisation energy of 70 eV, and Helium gas was used as the carrier gas at a constant flow rate of 1 mL/min.Mass analysis was performed by the regional centre for mycology and biotechnology, Al-Azhar University, Cairo, Egypt.General procedure for preparation of compounds 3a,b A solution of morpholine, 2a (0.871 g, 10 mmol) or piperidine, 2b (0.851 g, 10 mmol) in acetone was added dropwise over a period of 1 h to a stirred solution of cyanuric chloride 1 (1.845 g, 10 mmol) in 30 ml acetone at 0-5 C. Sodium carbonate solution was added after 4 h with vigorous stirring until pH reached 7-8.The slowly precipitated crystals were filtered off and dried 49,50 .

Biological evaluation
Procedure for in vitro antitumor screening in national cancer institute "NCI" The antitumor screening was performed for 60 human cancer cell lines under the protocol of NCI (www.dtp.nci.nih.gov), the Drug Evaluation Branch, Bethesda, MD.All submitted compounds were nominated based on the degree of structural diversity and computer modelling techniques for evaluation of their anticancer activity.The screening process for antineoplastic activity passes through two successive levels, starting with the assessment of target compounds at a single dose of 10 lM against 60 cancer cell lines on nine types of human tumours.These experiments were done on a triplicate following 54 .The output from the single-dose screening was covered as a mean graph, and then, analysed by the COMPARE program.Finally, the compounds which disclosed subpanel cancer cell growth (G %) equal to or less than 10% in at least eight cancer cell lines get to pass the assessment against the 60 cell lines at five doses (0.01-100 lM).By using GraphPad Prism version 9.0 (Graphpad Software Inc., La Jolla, CA), GI 50 , TGI, as well as LC 50 dose-response parameters were computed and presented for each compound (Supplementary information) [55][56][57] .

Assay of IDH2 enzyme inhibition
Assay of IDH2 R140Q enzyme inhibition was performed using the fluorimetry-based assay according to manufacturer's procedure of BPS Biosciences IDH2 (R140Q) Assay Kit, catalogue #79309 and wild type IDH Assay Kit, catalogue #71074.
In vitro cytotoxicity against human normal cells (human embryonic kidney) Assay of HEK-293 cell GI was performed as previously described by Tim Mosmann 58 .Data was measured in triplicate, and IC 50 values are given as mean values ± SD.

Assay of Annexin V-FITC apoptosis
Annexin V-fluorescein isothiocyanate (Annexin V-FITC) has high affinity to phosphatidylserine (PS).This affinity enables detection of PS by Annexin V-FITC staining as previously described 59 .
Cell cycle analysis HL-60(TB) cells were treated for 24 h with the GI 50 concentration of compound 6c.Following that, two runs of wash with ice-cold phosphate-buffered saline (PBS) were performed.And then, the treated HL-60(TB) cells were centrifuged and frozen in ethanol (70%, ice-cold) then washed in PBS, resuspended with RNase (100 mg/mL), stained with PI (40 mg/mL), finally analysed by flow cytometry using a FACS Calibur (Becton Dickinson, BD; Franklin Lakes, NJ).To calculate the cell cycle distributions, CellQuest software version 5.1 (Becton Dickinson) was used.

Western blot analysis
Compound 6c was incubated with the seeded cells following the procedure described by W. Neal Burnette 60 .

Molecular docking study
The crystal structure of IDH2 was downloaded as a PDB file from the research Collaboratory for structural bioinformatics (RCSB) Protein Data Bank (PDB ID: 5I96).The PDB file of the protein was opened in MOE version 2020.09 and all non-standard atoms and bonds were removed.Then the protein was prepared using the preparation options available in the program as well as energy minimised.Data base of the selected inhibitors was prepared by MOE.Docking was done and the results were visualised by Discovery Studio version 20.1.019295.

In silico physicochemical and pharmacokinetic study
We applied SwissADME model which is a free web tool developed by the Swiss Institute of Bioinformatics (SIB) (http://www.swissadme.ch) to predict physicochemical and pharmacokinetic properties of our compounds.A Boiled-Egg plot provides a supportive guidance and a statistical plot to foreshow the two passive prediction of small molecules, i.e. gastrointestinal absorption and brainpermeant.This model supplies a superior optimisation method.Gastrointestinal absorption and Brain access are two crucial pharmacokinetic parameters necessary in estimating the stages of the drug discovery processes.This computational tool also provides parameters; MLOGP, TPSA, and MW.Moreover, SwissADME tool confers a prediction to drug-likeness using the physicochemical properties and by applying number of rules as Lipinski, Ghose, Veber, Egan, and Muegge rules.

Figure 5 .
Figure 5. Mean growth inhibition percentage (GI %) on 60 cancer cell lines for series (I) and series (II) in the single dose experiment.

Figure 6 .
Figure 6.Summary of structure-activity relationship (SAR) of series (I), (II), and (III) as anticancer agents against 60 human subpanel cancer cell lines relying on the values of mean GI%.
Molecular docking studyDocking study was accomplished, by molecular operating environment (MOE 2020.09,Montreal, QC, Canada), to examine the probable types of binding modes for the most active analogues, 6c, 6e, 7c, and enasidenib on the allosteric site of the crystal structure of IDH2 R140Q (PDB: 5I96).Visualisation of compounds interactions was achieved via Discovery Studio (version 20.1.019295,San Diego) and MOE, where a high resolution (1.55 Ð) X-ray crystal structure of IDH2 R140Q mutant (PDB: 5I96) was selected24 .The

Figure 7 .
Figure 7. Effect of DMSO (upper two panels) and compound 6c (lower two panels) on the cell cycle distribution of HL-60(TB) cancer cell line.

Figure 9 .
Figure 9. Summary of the Annexin V-FITC Apoptosis assay results of compound 6c and DMSO on the percentage of HL-60(TB) cells stained positive for Annexin V-FITC.

Figure 10 .
Figure 10.Impact of compound 6c on expression of active Caspase 3 and Caspase 9 levels in HL-60(TB) cancer cells.

Figure 14 .
Figure 14.Radar charts for prediction of oral bioavailability profiles of compounds 6a, 6c, 6d, 7g, and 7l represented by red line, and the range of optimal property values are shown in pink.

Table 3 .
In vitro inhibition of mutant and wild type IDH2 enzymes by selected target compounds.
aThe mean ± SD of three experiments; b NT: not tested.

Table 4 .
Cytotoxic activity of compound 6c and staurosporine against the normal kidney cells of human embryo (HEK-293).

Table 5 .
Cell cycle analysis of HL-60(TB) cells treated with compound 6c and DMSO as a negative control.