Synthesis of N‐aminophalimides derived from α‐amino acids: Theoretical study to find them as HDAC8 inhibitors by docking simulations and in vitro assays

Phthalimides are valuable for synthesis and biological properties. New acetamides 3(a–c) and 4(a–c) were synthesized and characterized as precursors for novel N‐aminophalimides 5(a–c) and 6(a–c). Structures of 4a, 5(a–b), and 6(a–b) were confirmed by single crystal X‐ray. Docking studies identified compounds with favorable Gibbs free energy values for binding to histone deacetylase 8 (HDAC8), an enzyme targeted for anticancer drug development. These compounds bound to both the orthosteric and allosteric pockets of HDAC8, similar to Trichostatin A (TSA), an HDAC8 inhibitor. 6(a–c) contain hydroxyacetamide moiety as a zinc‐binding group, a phthalimide moiety as a capping group, and aminoacetamide moiety as a linker group, which are important for ligand‐receptor binding. ΔG values indicated that compounds 5b, 6b, and 6c had higher affinity for HDAC8 in the allosteric pocket compared to TSA. In vitro evaluation of inhibitory activities on HDAC8 revealed that compounds 3(a–c) and 5(a–c) showed similar inhibitory effects (IC50) ranging from 0.445 to 0.751 μM. Compounds 6(a–c) showed better affinity, with 6a (IC50 = 28 nM) and 6b (IC50 = 0.18 μM) showing potent inhibitory effects slightly lower than TSA (IC50 = 26 nM). These findings suggest that the studied compounds hold promise as potential candidates for further biological investigations.

On this background, our research has been interesting in the synthesis of compounds derived from αamino acids and in researching their biological properties, for instance, we have synthesized and carried out docking studies and in vitro assays of some 2,3-dihydro-1H-isoindole derived from αamino acids as channel blockers, Cox-1 and -2 inhibitors, effect over HDAC8 activity and expression, and HDACs inhibitors, as well as effect on the K14E6 transgenic mouse model (Mancilla et al., 2001;Mancilla-Percino et al., 2010, 2016;Rodríguez-Uribe et al., 2018, 2020;Santamaria-Herrera et al., 2016;Trejo Muñoz et al., 2014).Thus, continuing with our studies, in this work, we present the synthesis of novel N-aminophalimides 5(ac) and 6(a-c) derived from αamino acids, which have the phthalimide moiety as analogous compounds studied as anticancer agents and HDAC inhibitors, as well as new 3(a-c), 4(a-c) acetamides as precursors of Naminophalimides.The compounds under study were selected to their synthesis from molecular docking results of acetamides y N-aminophalimides derived of 20 αamino acids against HDAC8.All compounds synthetized in this work were characterized by nuclear magnetic and highresolution mass spectrometry, and confirmation of the structures of 4a, 5a, 5b, 6a, and 6b by X-ray diffraction.Furthermore, evaluation of the inhibitory activity of 3(ac), 5(a-c), and 6(a-c) on HDAC8 enzyme is reported.

| General procedures and materials
All solvents and chemicals were used as bought without further purification.HDAC8 Inhibitor Screening kit was bought from Sigma Aldrich Co® Catalog Number EPI007. 1 H and 13 C NMR spectra were recorded on a Jeol 500 ECA (500 MHz for 1 H; 125 MHz for 13 C), or BRUKER_DMX (400 MHz for 1 H and 100 MHz for 13 C) using Chloroformd, DMSO-d6, or Acetone-d6.Chemical shifts of 1 H and 13 C are reported relative to tetramethylsilane (TMS) and are given in parts per million (ppm).The following abbreviations were used for signal patterns: s (singlet), d (doublet), dd (doublet of doublets), dt (doublet of triplets), q (quarter), t (triplet), and m (multiplet).HRMS were recorded on an HPLC 1100 coupled to an MSD-TOF Agilent Technologies HR-MSTOF 1069A or Bruker micrOTOF-Q II spectrometer, both equipment with electrospray ionization (ESI).X-ray spectroscopy was performed using a Bruker D8 VEN-TURE or Bruker-APEX-II Diffractometer.IR spectra were recorded on a Varian FT-IR-IR 640 series.Melting points were determined on a Gallenkamp equipment MFB-595 with open capillary tubes and were uncorrected.Measurement of the enzymatic assay was Read Ex/Em = 390/485 nm using a Fluoroskan Ascent FL Thermo Scientific.

| Chemistry
2.2.1 | Synthesis of tert-Butoxy acetic acid (2)   In a 100 mL balloon flask equipped with a magnetic bar on an ice bath, NaH 60% dispersion in mineral oil (4 equiv., 2.40 g, 60 mmol) and anhydrous THF (50 mL) were added, anhydrous tert-butanol (4 equiv., 5.70 mL, 60 mmol) was added dropwise, the reaction mixture was allowed to stir for 20 min and Bromoacetic acid 1 (1 equiv., 2.09 g, 15 mmol) dissolved in anhydrous THF (10 mL) was added dropwise, The reaction mixture was allowed to warm to room temperature and subsequently heated to reflux for 6 h.After being cooled to room temperature, the solvent was removed under reduced pressure, the residue was dissolved in water (15 mL) and washed with hexane (10 mL × 3), the aqueous layer was neutralized with dilute HCl and extracted with dichloromethane (10 mL × 3), the organic layer was dried with magnesium sulfate anhydrous and filtered, and the solvent was removed under reduced pressure.The target compound was obtained as colorless oil 94% (1.86 g, 14.10 mmol).
2.2.2 | General procedure for the synthesis of compounds 3(a-c) In a 50 mL balloon flask provided with a magnetic bar on an ice bath, compound 2 (1 equiv., 4.5 mmol), the respective amino methyl ester hydrochloride (1 equiv., 4.5 mmol), dichloromethane (15 mL, for 3a) or acetonitrile (15 mL, for 3b and 3c) and DIPEA (3 equiv., 2.4 mL, 13.5 mmol) were added, the reaction mixture was allowed to stir for 20 min, T3P® ≥50 wt.% in ethyl acetate (1.2 equiv., 3.22 mL, 5.4 mmol) was added dropwise, the solution was stirred overnight at room temperature.The solvent was removed under reduced pressure, the residue was dissolved in dichloromethane (30 mL) and washed with distilled water and (10 mL), diluted HCl (10 mL), aq.NaHCO 3 (10 mL), and distilled water and (10 mL), the organic layer was dried with magnesium sulfate anhydrous and filtered, and the solvent was removed under reduced pressure.

| Computational methodology
Molecular docking of the acetamides and TSA (reference compound) was performed on the X-ray crystal structure of human HDAC8 enzyme, which was retrieved from RCSB Protein Data Bank (PDB ID: 1T64) (Somoza et al., 2004;Vannini et al., 2004).The crystal structure was optimized by removing the water and the co-crystallized ligands using Discovery Studio Visualizer v19.1.0.18287 (Dassault Systèmes BIOVIA, 2019), whereas hydrogen atoms were added using AutoDockTools-1.5.6.All the compounds were drawn using GaussView 5.0.8 and were geometrically optimized by density functional theory calculations at the B3LYP/6-31G** level using Gaussian 09W software (Frisch et al., 2013).Docking calculations were conducted using AutoDock 4.2.6®software (Morris et al., 2009).The grid box covered the entire volume of the HDAC8 (126 Å × 126 Å × 126 Å dimensions) with a grid spacing of 0.375 Å.The main Lamarckian Genetic Algorithm (Morris et al., 2009) parameters were set to 100 runs, a population size of 100, and a maximum number of 1 × 10 7 energy evaluation.The co-crystallized TSA was re-docked on HDAC8 to valid the docking protocol.The re-docked was evaluated by calculation of root standard deviation (RMSD) using Discovery Studio Visualizer v19.1.0.18287 (Dassault Systèmes BIOVIA, 2019).Docking poses of the target compounds and TSA were analyzed based on the best energy and were visualized using Discovery studio 3.5 software v19.1.0.18287 (Dassault Systèmes BIOVIA, 2019).

| HDAC8 inhibition assay
The inhibitory activity of 3(a-c), 5(a-c), and 6(a-c) on HDAC8 was evaluated by detection of lysyl deacetylase activity of the recombinant human HDAC8, using HDAC8 Fluorometric Drug Discovery Kit according to manufacturer's instructions (Sigma Aldrich Co® Catalog No EPI007).The stock solutions of the compounds and TSA (inhibitor control) were prepared in DMSO and diluted in assay buffer to reach the final concentration in the range of 0.25 nM-100 μM.The final concentration of DMSO was <10% and did not affect the activity of the assays.The assays were performed by preincubating the enzyme with the test compounds, including TSA, at 37°C for 10 min.Then, the substrate was added, and the mixture was incubated at 37°C for 60 min.After, 10 μL of the developer were added and the mixture was incubated at 37°C for 5 min.The fluorescence signal was measured using a fluorescence plate reader (Thermo Scientific Fluoroskan Ascent FL) at excitation and emission wavelengths of 390 and 485 nm, respectively.All experiments were performed in triplicate.The IC 50 values were calculated using nonlinear regression dose-response fit in (Prism GraphPad Software).

| Chemistry
The synthetic route leading to the formation of the target compounds 3(a-c), 4(a-c), 5(a-c), and 6(a-c) is outlined in Scheme 1. Compound 2, which was raw material, was obtained by the reaction of Bromo acetic acid 1 with tertbutanol in the presence of NaH.Compound 2 was converted by a coupling reaction to tert-butoxyacetamides methyl ester 3(a-c) with corresponding αamino methyl ester hydrochloride (a, b, and c: Gly, Phe, and Trp derived, respectively), using T3P® in DCM or ACN depending on their solubility.Compounds 3(a-c) were then converted into the corresponding tert-butoxyacetamides 4(a-c) after alkaline hydrolysis using potassium hydroxide in aqueous methanol.Compounds 5(a-c) were obtained through the coupling between N-aminophthalimide and corresponding 4(a-c), using HATU as coupling reagent with stirring at room temperature overnight.Finally, 6(a-c) were obtained by the deprotection of the hydroxyl group with Amberlite® IR120 hydrogen form under methanol reflux (Mallesha et al., 2012).

| Spectroscopy
All compounds were characterized by spectroscopic methods of 1 H, 13 C NMR (Spectra are given in Appendix S1), Infrared and spectrometry of HRMS (Spectra are given in Appendix S1).Besides, suitable crystals of compounds 4a, 5a, 5b, 6a, and 6b were obtained for X-ray diffraction, which confirmed their structures (X-ray data are given in SI).All data for each compound are given in the materials and methods section. 1 H NMR spectra of compounds 3a, 3b, 4a, and 4b were recorded in CDCl 3 , while 3c and 4c in DMSO-d6.The spectra of 3(a-c) showed a singlet signal for methoxy and tert-butoxy protons in the range of 3.62-3.77and 1.06-1.25 ppm, respectively.H-2 of 3a displayed a doublet at 4.09 ppm, and that of 3b and 3c showed a quartet at 4.89 and 4.61 ppm, respectively.The NH-3 of 3a exhibited a singlet signal at 7.18 ppm and that of 3b and 3c a doublet at 7.08 and 7.45 ppm, respectively.The H-5 showed a singlet signal between 3.75 and 3.94 ppm.Benzyl protons of 3b and indolyl group of 3c shown the coupling patterns expected in the range of 3.14-3.22ppm for H-12 and between 6.98 and 7.47 ppm for aromatics protons and NH of indolyl group showed a singlet at 10.96 ppm. 1 H NMR spectra of 4(a-c) showed for tert-butoxy, H-2, NH-3, benzyl of 4b, and indolyl of 4c protons almost the same chemical shifts and coupling patterns observed for 3(a-c).The carboxylic protons displayed a broad signal in the range of 8.91 and 12.84 ppm.H-5 of 4a and 4c exhibited a singlet signal at 3.97 and 3.73, respectively, while those of 4b exhibited an AB system at 3.87 and 3.89 ppm.
H-5 of 5a exhibited a simple signal at 3.82 ppm, and those of 5b and 5c exhibited an AB system between 3.75 and 3.90 ppm.H-10 and H-11 of phthalimide moiety displayed a complex pattern in the range of 7.71-7.97ppm, NH-6 showed a singlet in the range of 9.07-10.75ppm.Spectra of 6(a-c) showed for H-2 of 6a a doublet at 4.00 ppm, while that of 6b and 6c exhibited a complex pattern at

| MOLECULAR DOCKING STUDY
X-ray structures of various HDAC8-inhibitor complexes have been described, including TSA among other (Brunsteiner & Petukhov, 2012), and considerable structural differences were found in the protein surface in the vicinity of the aperture of the active site, depending on which inhibitor is bonded (Estiu et al., 2010), these changes derived from the presence of two deep pockets one denoted as the orthosteric pocket and the second close to the first denoted as the allosteric pocket, where Trp141, Phe152, and Tyr306 form the wall between both cavities (Estiu et al., 2010).In particular, the crystal structure of HDAC8-TSA complex (PDB ID: 1T64) (Somoza et al., 2004), shows that TSA binds at two pockets, in this sense, it has been considered as competitive inhibitor because it is binding at catalytic pocket and noncompetitive inhibitor since is noncatalytic binding pocket (Su et al., 2008).The orthosteric that is the active site is described as a long and narrow tunnel constituted for the amino acids residues His142, His143, Gly151, Phe152, His180, Phe208, Met274, and Tyr306 (Somoza et al., 2004;Vannini et al., 2004), at the bottom of this tunnel, the catalytic site is found, which is formed by Zn 2+ ion and the catalytic triad of His142, His143, and Tyr306n (Gantt et al., 2016).One of the characteristics in HDAC8 is loop L1, which is formed by seven amino acid residues (Ser30, Leu31, Ala32, Lys33, Ile34, Pro35, and Lys36) (Maolanon et al., 2016), is highly flexible and found toward the proximity of the active site and can undergo conformational changes for substrates binding.
F I G U R E 1 Molecular structure of C 8 H 15 NO 4 , and crystallographic numbering scheme of 4a.
Thus, in this work, we described the binding mode of 3(a-c), 4(a-c), 5(a-c), and 6(a-c), as well as for TSA on two pockets of X-ray structure of HDAC8 (PDB ID: 1T64).Hence, a molecular docking study was performed to investigate binding modes of compounds 3(a-t), 4(at), 5(a-t), and 6(a-t) derived of 20 αamino acids (Gly, Phe, Trp, Ala, Val, Leu, Ile, Met, Tyr, Ser, Pro, Thr, Cis, Asn, Gln, Lys, His, Arg, Asp, and Glu) on HDAC8; results showed that most of the compounds binding in both orthosteric and allosteric site of the enzyme, as it has been observed for TSA (Docking data are given in SI).Based on the results, compounds 3(a-c), 4(a-t), 5(a-t), and 6(a-t) were selected for analysis of the binding modes on the X-ray structure of HDAC8 (PDB ID: 1T64).The docking protocol was confirmed through the re-docking of the co-crystallized TSA within the HDAC8 structure.It showed that the inhibitor bound into both pockets of the enzyme providing an RMSD value of 0.9304 and 1.3509 Å.Therefore, the docking protocol was reliable for the analysis of the binding modes of the target compounds with HDAC8.The Gibbs free energies (ΔG, kcal/mol), along with the corresponding dissociation constants (K d , μM) values were obtained for the four series HDAC8-ligands complexes in each pocket, showing all these compounds more affinity at the allosteric pocket than in orthosteric pocket, except 5c, and 4a that did not show affinity to orthosteric site.Furthermore, compounds 5b, 6b, and 6c inhibited more affinity than TSA.The data are summarized in Table 4.
The study of binding modes of 3(a-c), 4(a-c), 5(ac), and 6(a-c) and TSA with amino acid residues of both pockets of HDAC8 was based on the analysis of the type of interactions, such as hydrogen bonds, π-π interactions coordination bonds, πsulfur, πlone pairs, and πcation interactions, where the analysis of the distances were considered as <4 Å for hydrogen bonds and <6 Å for hydrophobic interactions, (Brylinski, 2018;Copeland, 2000) between heteroatoms, carbons, or protons coupled to heteroatoms of all target acetamides to atoms of amino acids residues of HDAC8.The docking results showed that all the binding interactions depended on the conformations and functional groups of the acetamides.

with HDAC8
Acetamides 3(a-c) showed interactions in the orthosteric site of HDAC8, in which binding modes depended on the orientation of the molecules based on the substitute in position three.Thus, the oxygen atom of carbonyl-1 of ester moiety of 3a showed a coordination bond to Zn 2+ at 2.22 Å, by hydrogen bonds with the proton of OH (side chain) of Tyr306 (2.23 Å) and with the NH (backbone) of Gly304 (2.51 Å).A carbon atom from the tert-butoxy moiety showed hydrophobic binds alkyl-π (side chain) of Phe208 (3.71 Å) and Phe152 (4.23 Å).Acetamides 3b and 3c, which have the phenyl and indole groups at position three, respectively, showed that both groups interacted with the amino acid residues of the surface of the enzyme in the vicinity of the opening of the active site, exhibiting different orientation, and bind modes to that observed for 3a, which does not have a substituent at position three.This can be attributed to the different conformations adopted by the three acetamides in the pocket.Thus, the oxygen atom of tert-butoxy group of 3b exhibited a hydrogen bond with the proton of NH (backbone) of Phe208 at 2.12 Å, phenyl group exhibited π-π T-shaped interaction with Phe152 at 4.89 Å, and πlone pair interaction (side chain) with His180 at 4.09 Å. Hydrogens, NH-3 and NH of indole group of 3c showed hydrogen bonds with the oxygen of carbonyl (backbone) of Gly206 (1.99 Å) and carbonyl (side chain) of Asp101 (2.09 Å), respectively; indole exhibited π-π stacking interactions with Phe208 at 3.82 and 3.94 Å, and πsulfur interaction with Met274 at 5.18 Å.The results show that compounds 3(a-c) bonded to the residues of the active site of HDAC8, and 3a additionally bonded to zinc ion, suggesting it bonded in a competitive manner.Compounds 3b and 3c also showed interactions with the amino acid residues that do not belong to the active site, which are in the L8 loop as Tyr306, the L5 loop as Phe 208, and Asp101 residue is strictly conserved in the L2 loop of all class I and class II HDAC's and has been established that is important for the substrate recognition (Dowling et al., 2008;Estiu et al., 2010;Vannini et al., 2007).The results showed that the ester group of 3a oriented toward the catalytic site as the hydroxamic group of TSA.Also, acetamides and inhibitor showed interactions with the same amino residues of the active site Phe152, Phe208, Gly304, Met274, and Tyr306.Therefore, acetamides 3(a-c) may be considered as possible HDAC8 inhibitors.
Acetamides 3(a-c) exhibited different conformations between them at the allosteric site of HDAC8, therefore displayed the following binds, 3a showed four hydrogen bonds, NH-3 with C=O (backbone) of Cys28 at 2.79 Å, with C=O (side chain) of Asp29 at 2.21 Å and the oxygen atom of the C=O-4 with NH 2 of guanidine group (side chain) of Arg37 at 1.88 Å.A carbon atom from tert-butoxy moiety showed alkyl-π interaction with Tyr306 at 5.05 Å. Compound 3b showed two hydrogen bonds, the oxygen atom of C=O-1 with NH (backbone) of Arg37 at 2.15 Å and NH-3 with OH (side chain) of Tyr111 at 2.50 Å, and the phenyl group exhibited π-π T-shaped interaction with Tyr 306 at 5.03 Å. Indole moiety of 3c showed πalkyl interaction with C (side chain) of Arg37 at 4.97 Å and πguanidine group protonated interaction (side chain) at 4.45 Å. NH of indole moiety exhibited a hydrogen bond with OH (side chain) of Tyr111 at 1.88 Å, also indole group showed π-π stacked interaction with Trp141 at 4.38 Å.Among the common binding of these compounds are to Arg37 through C=O-4 and C=O-1 of 3a and 3b, respectively, and indole group of 3c, acetamides 3a and 3d bind to Tyr306 through tert-butoxy moiety and phenyl moiety, respectively; 3b and 3c bind to Tyr111 by NH-3 and NH of indole moiety, (Figure 4).These results revealed that both acetamides and TSA bind to the amino acid residues Arg37, Tyr111, and Ser138 in the allosteric site of HDAC8, but different binding types depending on their conformation adopted at the site.

with HDAC8
Compounds 4(a-c), which have a carboxylic group instead of the ester group of their precursors 3(a-c), showed different orientations and binding types in the orthosteric site in comparison with those of 3(a-c), except 4a that did not show interactions in this pocket.Thus, the tert-butoxy group of 4b and 4c orient toward the inside of the tunnel, while the groups of 3b and 3c oriented inside were phenyl and indole, respectively.Two carbon atoms of the tertbutoxy moiety of 4b exhibited alkyl-π interactions with side chains of His143, Phe152, and His180 at 3.89, 4.44, and 4.12 Å, respectively, and two carbon atoms of the tertbutoxy group of 4c displayed alkyl-π interactions with the side chain of Phe152 (4.20, 4.60 Å), His180 (4.44 Å), Tyr306 (4.18 Å), and Met274 at (4.59 Å).C=O-1 of 4b and 4c bind by a hydrogen bond to NH (backbone) of Phe208 at 1.88 and 1.79 Å, respectively.The hydrogen atom of the carboxylic acid of 4b exhibited hydrogen bond with N (side chain) of His180 at 2.24 Å, NH-3 of 4b and 4c displayed hydrogen bonds with N (side chain) of His180 at 2.72 and 2.14 Å, respectively.C-5 of 4c exhibited alkyl-π interaction with the side chain of Phe208 at 3.70 Å. NH of indole moiety of 4c bind by a hydrogen bond to C=O (backbone) of Pro273 at 2.01 Å, although it does not belong to active site residues, it is joined to Met274, and indole moiety exhibited bind πalkyl interaction with C (side chain) of Met274 at 4.17 Å.These data showed that 4b and 4c interacted to the amino acid residues of the active site of HDAC8 and that carboxylic acid moiety contributed to the orientation of the molecules where tert-butoxy moiety, although voluminous, was oriented in the tunnel.These two acetamides and inhibitor showed binding with the same amino residues of the active site Phe152, Phe208, Met274, and Tyr306, where 4c displayed more bindings.Therefore, 4(b-c) may be considered HDAC8 inhibitors.
Compounds 4(a-c) in the allosteric site showed the following binding, for 4a, hydrogen bonds HO of the carboxylic acid with OH (side chain) of Ser138 at 2.18 Å, C=O-1 with guanidine group (side chain) of Arg37 at 1.63 Å, and with NH-3 (backbone) of Cys28 at 2.15 Å, and hydrophobic interactions alkyl-π interactions between carbon atoms of tert-butoxy moiety and (side chain) of Trp141 and Tyr306 at 4.89 and 4.94 Å, respectively.4b exhibited the following hydrogen bonds, the proton of carboxylic acid moiety with C=O (backbone) of Cys28 and C=O (side chain) of Asp29 at 2.89 and 1.80 Å, respectively, NH-3 with (backbone) of Pro35 at 2.5 Å, and phenyl moiety showed π-π T-shaped interaction with (side chain) of Trp141 at 4.77 Å. 4c displayed hydrogen bonds between NH-3 with C=O (backbone) of Pro35 at 2.15 Å, NH of indole group with OH (side chain) of Trp141 at 1.80 Å, and hydrophobic πsigma interaction was observed between indole moiety and (side chain) of Ile34 at 3.58 Å. Carbons of tert-butoxy moiety exhibited alkyl interaction with C (side chain) of Arg37 at 3.99 Å, and Trp141 at 3.99 and 4.46 Å, respectively.These acetamides showed binding with Try141, which is one of the residues that form the wall between the two pockets, they also show inhibitor shown interactions with the same amino acid residues Pro35, Arg37, Ser138, and Tyr111 (Figure 5).

with HDAC8
Compounds 5(a-c), which have N-aminophthalimide group showed differences in the orientation of the compounds toward the orthosteric site, phthalimide group of 5a and 5c was orient to the pocket favoring hydrophobic interactions and with the residues that they form the walls of the channel, while 5b maintains the orientation of the tert-butyl group toward the pocket, leaving the indole part on the surface, which allows greater interaction with surface residues and aromatic groups of the amino acids residues.The types of binding are described below.
The oxygen atom of C=O-1 and NH-3 of 5a bind by hydrogen bonds with NH (backbone) of Phe208 and the oxygen of Gly206 at 1.86 and 2.15 Å, respectively, π-π T-shaped interactions were between phthalimide moiety and the side chain of His180 and Phe208 at 4.69 and 4.12 Å, also πsulfur interaction between phthalimide and Met274 at 5.06 Å was observed.Compound 5b only showed πalkyl interactions between phthalimide moiety and the side chain of Pro35 at 5.46 Å, and π-π T-shaped interactions with the side chain of Phe152 and Tyr306 at 4.45 and 5.38 Å. Carbon atoms of the tert-butoxy group πalkyl interactions with His143, His180, and Phe208 were displayed at 4.71, 3.59, and 4.23 Å, respectively, also phenyl moiety presented πsulfur interaction with Met274 at 4.47 Å. NH of indole moiety of 5c showed a F I G U R E 4 Binding modes of HDAC8-3(a-c): 3a-, 3b-, and 3c-O represent binding of the compounds with the amino acid residues in the orthosteric site; 3a-, 3b-, and 3c-A represent the binding in the allosteric site.Bindings were hydrogen bonds (green dotted line), hydrophobics π-π (purple dotted line), πalkyl (pink dotted line), πsulfur and πguanidine group protonated (orange dotted line), and Zn ion (orange ball).
hydrogen bond with the backbone of Gly206 at 1.97 Å, and carbon atoms of tert-butoxy moiety exhibited πalkyl interactions with the side chain of Phe207 and Met274 at 4.62 and 4.16 Å, respectively.These results showed that although no compound showed the suitable conformation to bind the zinc ion, they and TSA displayed interactions with the same amino acid residues of the active site Phe152, Phe208, Met274, and Tyr306.Besides, phenyl moiety of TSA showed binding only with Met274, and the phthalimide group of 5a and 5b displayed binding with His180, Phe208, Met274, and Phe208, Tyr306, respectively, while that of 5c did not show bind.Therefore, 5(a-c) could be considered as possible HDAC8 inhibitors.
Compounds 5(a-c) in the allosteric site showed the following binds, phthalimide moiety of 5a exhibited π-π T-shaped interactions with the side chain of Phe208 (4.51 and 4.75 Å), 5b with the side chain of Tyr306 at 5.09 Å, and πalkyl interactions of these compounds with the side chain of Pro35 (3.72, 4.0, and 3.63 Å).While phthalimide moiety of 5c displayed πalkyl interactions with the side chain of Ile34 and Pro35 at 4.96 and 4.50 Å, respectively.NH-3 and oxygen atom of the tert-butoxy moiety of 5a showed hydrogen bonds with OH of the side chain of Tyr111 at 2.11 and 2.27 Å.The carbon atoms of the tertbutoxy moiety of 5a exhibited πalkyl interactions whit the side chain of Trp141 at 4.86 and 5.03 Å, 5b with the side chain of Tyr111 at 3.95 and 5.05 Å and the side chain of Trp141 at 3.80 and 4.56 Å, and 5c with the side chain of Tyr100 at 4.46 and 5.18 Å. Phenyl moiety of 5b showed πguanidine group protonated interaction (side chain) of Arg37 at 3.97 Å, and indole moiety of 5c exhibited π-π Tshaped interaction with the side chain of Tyr100 at 5.08 Å (Figure 6).The data showed that phthalimide moiety of 5(a-c) showed binding with Pro35, Arg37, Ile34, Phe208, and Tyr306, while phenyl moiety of TSA showed binding with Pro35 and Phe152.5a and 5b conformations allowed to these binding with the inner amino acid residues of the pocket (Tyr111,Trp141,Phe208,and Tyr306), where these compounds and TSA showed binding with Tyr111, while

with HDAC8
It is known that several classes of small molecule have been recognized as HDAC inhibitors, most of these are hydroxamic acid derivatives, exemplified by suberoylanilide hydroxamic acid (SAHA), where hydroxamate moiety chelate the zinc ion in the active site.While hydroxamic acid group is responsible for various potent inhibitors, they usually have many problems associated with their use such as low oral availability, poor in vivo stability, and undesirable side effects.Based on the above, research on this topic was focused on the replacement of hydroxamate by non-hydroxamate groups to find potent molecules as inhibitors of HDACs, thus, several suberoylanilide hydroxamic acid (SAHA)-based compounds have been synthesized containing sulfonamide, sulfone, aminocatetamide, hydroxyacetamine, and mercaptoacetamide as ZBG.The kinetic enzyme assays and molecular modeling suggested that compounds with mercaptoacetamide moiety interacts with the zinc in the active site of HDACs (Suzuki et al., 2005;Wang & Dymock, 2009).
In this context, compounds 6(a-c) are attractive because having a hydroxyacetamide moiety as a zincbinding group (ZBG), a phthalimide moiety as a capping group, and aminoacetamide moiety as a linker group, which are important in the ligand-receptor binding (Marks et al., 2003; Figure 7).The binding modes of these compounds with HDAC8 depend on their conformations led to different orientations on each site.Hydroxyl group F I G U R E 6 Binding modes of HDAC8-5(a-c): 5a-, 5b-, and 5c-O represent binds of the compounds with the amino acid residues in the orthosteric site; 5a-, 5b-, and 5c-A represent the binds in the allosteric site.Bindings were hydrogen bonds (green dotted line), hydrophobics π-π (purple dotted line), πalkyl (pink dotted line), and πsulfur and πguanidine group protonated (orange dotted line).

F I G U R E 7
Structure general for 6(a-c), red color ZBG, green color LG, and blue color CG.
of 6a was orient toward the inner of the orthosteric site, where the oxygen of C=O-4 showed binding at zinc ion at 2.05 Å.Also, 6a exhibited hydrogen bonds with the enzyme as follows, hydrogen of NH-3 with oxygen atom (backbone) of Gly151 at 2.5 Å, oxygen of C=O-4 with OH (side chain) of Tyr306 at 2.22 Å, hydrogen of NH with oxygen atom (backbone) of Gly304 at 2.61 Å, and hydrogen of OH group bind to an oxygen atom (side chain) of Asp178 at 2.10 Å. Phthalimide moiety showed π-π stacking interactions with Phe208 at 3.83 and 4.44 Å. Due to 6a binding to zinc ion and Asp178, Phe208, and Tyr306 just like TSA, it may be promising as an HDAC8 inhibitor, and a candidate for additional studies.
Compounds 6b and 6c were oriented at the active site in a different way than 6a because they had different conformations.Although these compounds did not show interaction with the zinc ion, they showed binding interactions with the amino acid residues of the active site.Compound 6b displayed interactions by hydrogen bonds as follows, hydrogen of NH-3 and that of hydroxyl group interacted with oxygen (side chain) of Asp101 at 2.08 and 1.97 Å, respectively, the oxygen atom of C=O-8 with NH (backbone) of Phe208 at 1.97 Å.The aromatic moiety of phthalimide of 6b exhibited π-π T-shaped interaction whit (side chain) of His180 at 5.67 Å and πalkyl with (side chain) of Met274 at 4.38 and 4.60 Å. Compound 6c exhibited interactions by hydrogen bonds as follows: Oxygen atom of C=O-4 with the hydrogen of NH (backbone) of Phe208 at 1.69 Å, hydrogen of OH group with oxygen (side chain) of Gly206 at 1.77 Å, NH-6 with S (side chain) of Met274 at 2.98 Å, and the oxygen atom of C=O-8 with NH (side chain) Lys202 at 2.20 Å.The phthalimide moiety showed πalkyl interaction with (side chain) of Met274 at 5.02 Å, indole moiety showed the π-π T-shaped interactions with Phe152 (4.78 Å) and His180 (4.31 Å), and the π-π stacked with Phe208 at 4.13 and 4.86 Å.As it is mentioned above, although 6b and 6c did not interact with zinc ion, they showed binding interactions with the amino acid residues of the active site Phe208 and Met274 as well as TSA; therefore, these compounds could be considered for further studies, (Figure 5).Compounds 6(a-c) in the allosteric site showed hydrogen bonds interactions of the oxygen atoms of C=O-1 and C=O-4 of 6a with guanidine moiety (side chain) of Arg37 at 2.66 and 2.49 Å, respectively, whereas only oxygen atom of C=O-4 of 6b bind to Arg37 at 1.83 Å, NH-6 of 6a and 6b with oxygen (backbone) of Pro35 at 1.78 and 2.18 Å, respectively, oxygen of OH group of 6a with OH (side chain) of Tyr111 and hydrogen of HO with of 6a (side chain) of Ser138 at 1.80 and 2.10 Å, hydrogen of OH group of 6b with oxygen (backbone) of Ser138 at 1.66 Å, and hydrogen of OH group of 6c whit OH (side chain) of Tyr111 and oxygen (backbone) of Phe152 at 1.95 and 2.07 Å.
The phthalimide moiety displayed the following interactions: For that of 6a πalkyl with CH 2 (side chain) of Pro35 (4.13 Å), CH 2 (side chain) of Arg37 (5.15 Å), and π-π T-shaped (side chain) of Phe152 at 4.82 Å, for 6b and 6c a π-π T-shaped with the side chain of Tyr306 at 4.54 and 4.53 Å, respectively.Phenyl moiety of 6b displayed π-π stacking interaction with the side chain of Tyr111 at 4.61 Å.The hydrogen of NH of indole moiety of 6c showed a hydrogen bond with oxygen (backbone) of Pro35 at 1.75 Å, and the indole moiety exhibited π-π T-shaped interaction with (side chain) of Tyr111 at 5.91 (Figure 8).The results showed that phthalimide moiety of 6a bound with Ile34, Pro35, and Phe152, and that of 6b and 6c with Tyr306, while phenyl moiety of TSA bind with Pro35 and Phe152.The hydroxy group of these compounds bind with Tyr111 (6a, 6c), Ser138 (6a, 6b), and Phe152 (6c), while the hydroxamic moiety of TSA did not display interaction in this pocket, but the inhibitor showed interactions with the same amino acid residues by binding mode different to those of the target compounds.Thus, compounds 6(a-c) are promising for further studies.

| HDAC8 INHIBITORY ASSAY
It is known that several drugs have been identified to inhibit class I and/or class II histone deacetylases (HDACs), of which Trichostatin A (TSA) is one of the well-known HDAC inhibitors.However, little is known about the effect of TSA on HDAC8, of which is the inhibition of proliferation of Molt-4 cells which express HDAC8 (Jing et al., 2006).Another study has been conducted to explore whether the function of HDACs was modulated by TSA at concentrations that block cancer cell viability by enzyme activity assays in purified systems and in cell extracts.TSA's effective killing dose of 400 nM fully inhibited HDAC1 and HDAC2, and HDAC8 only partly activity, however, 5 mM TSA fully eliminated its activity (Chang et al., 2012).
The inhibitory activity of compounds 3(a-c), 5(a-c), and 6(a-c) on HDAC8 was evaluated based on the results obtained from the docking study because they presented better values of ΔG and K d than 4(a-c), and even 4a did not present interactions in the orthosteric site of HDAC8.The in vitro inhibitory activity of target acetamides was evaluated with the HDAC8 Fluorometric Drug Discovery Kit, TSA as a control inhibitor included in the Kit.Table 5 shows the IC 50 values, although 5(a-c) showed better values of ΔG and K d from the docking study than 3(a-c), both series exhibited almost the same inhibitory effect in the range of 0.751-0.445μM.Regarding compounds 6(a-c), that displayed better affinity in docking, 6a (IC 50 of 28 nM) and 6b (IC 50 = 0.18 μM) revealed the best effect inhibitory effect on HDAC8, except for 6c.Compound 6a showed the best IC 50 slightly above the TSA.

| CONCLUSION
Twelve new acetamides and novel N-aminophalimides derived from αamino acids were selected for their synthesis from docking study and were obtained in good yields; they were characterized by 1 H and 13 C NMR, infrared, HRMS, and structures of 4a, 5a, 5b, 6a, and 6b were confirmed by X-ray crystallography.The molecular docking results showed that all compounds, except 4a binds in both pockets, and their orientation within HDAC8 depended on the conformation in each site.In general, acetamides without and with phthalimide moiety showed interactions with the amino acid residues of the active site and allosteric site, where binding modes were similar or different to those displayed for TSA.Although only 3a and 6a displayed interaction with the Zn, all target acetamides studied may be considered for further studies.According to the inhibition assays, all the evaluated acetamides exhibited inhibitory activity on HDAC8.Among them, 6a (IC 50 = 28 nM) and 6b showed better enzymatic inhibitory, where 6a showed the greatest effect, slightly less than TSA (IC 50 = 26 nM).These results suggested that the target acetamides and N-aminophalimides may be promising anticancer agents for further biological studies.F I G U R E 8 Binding modes of HDAC8-6(a-c): 6a-, 6b-, and 6c-O represent binds of the compounds with the amino acid residues in the orthosteric site; 6a-, 6b-, and 6c-A represent the binds in the allosteric site.Bindings were hydrogen bonding (green dotted line), hydrophobics π-π (purple dotted line), πalkyl (pink dotted line), and interactions πsulfur (orange dotted line).The zinc ion is represented by an orange ball. 2 5.06 and 4.89 ppm, respectively.H-5 of 6a exhibited a doublet signal at 3.86 ppm by coupling with OH, which showed a triplet at 5.57 ppm, whereas H-5 and OH of 6b displayed a simple signal at 3.95 and 4.64, respectively, H-5 of 6c showed a complex pattern at 3.74 ppm by coupling with OH, which showed a triplet at 5.48 ppm.NH-3 of 6a showed a triplet at 8.09 ppm, while that of 6b and 6c exhibited a doublet at 7.55 and 7.70 ppm, respectively.NH-6 for these compounds displayed a simple signal in the range of 10.02-11.08 ppm.H-10 and H-11 of 6a and 6c exhibited a complex pattern at 7.94 ppm, while those of 6b showed a simple signal at 7.95 ppm.H-12 of 6b and 6c displayed an AB coupling system in the range of 3.18-3.33ppm, chemical shifts of the aromatic protons of benzyl and indolyl groups are in the range of 6.96-7.61ppm, and NH of indolyl showed a simple signal at 10.90 ppm. 13C NMR spectra of 3(a-c) and 4(a-c) showed a signal for methyl of the tert-butoxy groups in the range of 27.0-27.4ppm, for carbon the CH 3 O group of 3(a-c) the signal at 52.3 ppm, C-2 showed the signal between 40.5 and 52.5 ppm, and C-5 between 61.7 and 62.0 ppm.The signal of quaternary carbon of the tert-butoxy group was in the range of 74.0-74.9ppm, the C-1 of the compounds 3(a-c) showed the signal between 170.2 and 172.0 ppm, whereas C-1 of the compounds 4(a-c) exhibited the signal within 172.4-174.3ppm, C-4 exhibited the signal within 169.8-172.3ppm.Carbons of the benzyl and indolyl groups S C H E M E 1 Synthetic rout 3(a-c), 4(a-c), 5(a-c), and 6(a-c).Reagents and conditions: (a) NaH, t-BuOH, THF 0°C to rt, reflux; (b) respective αamino methyl ester hydrochloride (a, b, and c: Gly, Phe, and Trp derived, respectively), DIPEA, T3P®, DCM, or ACN 0°C to rt; (c) KOH, MeOH:H 2 O; (d) N-aminophthalimide, HATU, DIPEA, DMF; (e) Amberlite® IR120 hydrogen form, MeOH, reflux.displayedsignals between 26.7 and 38.0 ppm for C-12, in the range of 127.2-135.8ppm for C-13 to C-16, and within 108.8-136.2ppm for C-13 to C-20. 13 C NMR spectra of 5(a-c) and 6(a-c) showed that chemical shifts of methyl carbons of the tert-butoxy moiety of 5a (27.0-27.2ppm), C-2 (39.5-52.0ppm), C-5 (61.3-61.9ppm), quaternary carbons of the tert-butoxy groups of 5a (73.9-74.8ppm), C-1 (168.5-171.5 ppm),C-4 (170.6-172.6 ppm), C-12 (27.8- 37.9), C-13 to C-16 (126.6-136.7 ppm), and C-13 to C-20  (109.4-136.6)were almost identical to that of 3(a-c) and 4(a-c).Aromatic carbons C-9 to C-11 and carbonyl carbons C-8 of the phthalimide moiety displayed the signals in the range of 123.5-135.9ppm and 164.7-165.6 ppm, respectively.The high-resolution mass spectra of the target compounds showed the ion of the protonated molecules [M + H] + , which agreed with their molecular formula, the data are given in the experimental section.

F
Molecular structure of C 16 H 19 N 3 O 5 and C 24 H 26 Cl 3 N 3 O 5 , crystallographic numbering scheme of 5a and 5b, respectively.F I G U R E 3 Molecular structure of C 12 H 11 N 3 O 5 and C 19 H 17 N 3 O 5 , crystallographic numbering scheme of molecules 6a and 6b, Bond lengths selected (Å) for 5a and 5b.

F
Binding modes of HDAC8-4(a-c): 4b-O and 4c-O represent binds of the compounds with the amino acid residues in the orthosteric site; 4a-, 4b-, and 4c-A represent the binding in the allosteric site.Bindings were hydrogen bonds (green dotted line), hydrophobics π-π (purple dotted line) and πalkyl (pink dotted line), and πsulfur (orange dotted line).5c conformation let more binding with amino acid residues of the surface pocket.
aValue is given in nM.