Phenylacetyl-/Trolox- Amides: Synthesis, Sigma-1, HDAC-6, and Antioxidant Activities

In search of novel multi-mechanistic approaches for treating Alzheimer’s disease (AD), we have embarked on synthesizing single small molecules for probing contributory roles of the following combined disease targets: sigma-1 (σ-1), class IIb histone deacetylase-6 (HDAC-6), and oxidative stress (OS). Herein, we report the synthesis and partial evaluation of 20 amides (i.e., phenylacetic and Trolox or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid derivatives). Target compounds were conveniently synthesized via amidation by either directly reacting acyl chlorides with amines or condensing acids with amines in the presence of coupling agents 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (HATU) or 1,1′-carbonyldiimidazole (CDI). Overall, this project afforded compound 8 as a promising lead with σ-1 affinity (Ki = 2.1 μM), HDAC-6 (IC50 = 17 nM), and antioxidant (1.92 Trolox antioxidant equivalents or TEs) activities for optimization in ensuing structure–activity relationship (SAR) studies.


Introduction
Alzheimer's disease (AD) is a progressive poly-factorial brain pathology in which σ-1, HDAC-6 activity, and oxidative stress (OS) have intertwined or synergistic neurodegenerative contributory roles [1][2][3][4].We opine that single small molecules armed with combo σ-1 stimulatory and HDAC-6 and antioxidant or OS inhibitory properties could offer a novel multi-mechanistic approach for developing anti-neurodegenerative and potentially disease-modifying agents for AD.
HDAC-6 is a class IIb α-tubulin hydrolytic enzyme which removes acetyl moieties from N-acetylated lysine groups of histone and nonhistone proteins (e.g., peroxiredoxin enzymes) [10].Due to its role as a regulatory protein, the HDAC-6 isoform has attracted much interest for druggability in multiple diseases, including CNS disorders.A sampling of evidence in support of this enzyme's role in AD includes elevated HDAC-6 protein levels in AD brains (especially in the cortical and hippocampal regions) versus normal aged comparators, diminished tau phosphorylation in both HDAC-6 inhibition and knockdown studies, and cognitive improvement observations in cross studies of HDAC-6 knockdown versus amyloid beta (Aβ)-induced memory deficit models.Taken together, HDAC-6 has a multi-pronged contributory role in neurodegeneration and is a validated druggable target for AD [11].
With regard to OS, in general, excessive radical/nonradical reactive species coupled with diminished brain antioxidant enzyme capacity have been linked to neurodegeneration via a variety of mechanisms (lipid peroxidations, Ca 2+ excitotoxicity, mitochondrial dysfunction, etc.).Additionally, OS also promotes higher HDAC-6 enzyme expression [2,3].OS-induced alterations, therefore, often culminate in synapse dysfunction, which correlates well with cognitive declines in AD patients [4].The aberrantly produced Aβ oligomers also induce reactive oxygen species (ROS) production, leading to neuronal apoptosis [12].Exemplified by glycogen synthase kinase-3 (GSK3) studies, OS can also promote tau hyperphosphorylation, leading to intraneuronal accumulations of neurofibrillary tangles (NFTs) and eventual neuronal death [12].Interestingly, both Aβ and hyperphosphorylated tau beget more OS and the cycle continues.Although antioxidants have performed poorly in clinical trials, there is still discovery space for brain penetrant lipophilic ones, which could potentially minimize lipid peroxidation in neurodegenerative diseases [13].
Since our ultimate goal is to develop single molecules capable of activating σ-1 while inhibiting both HDAC-6 and OS, we designed our compounds to incorporate three key pharmacophoric elements (i.e., two lipophilic groups (Cap or LG 1 and LG 2 ) plus a linker) common to both σ-1 agonists [14] and HDAC-6 inhibitors [15] (see Figure 1).
enzymes) [10].Due to its role as a regulatory protein, the HDAC-6 isoform has attracted much interest for druggability in multiple diseases, including CNS disorders.A sampling of evidence in support of this enzyme's role in AD includes elevated HDAC-6 protein levels in AD brains (especially in the cortical and hippocampal regions) versus normal aged comparators, diminished tau phosphorylation in both HDAC-6 inhibition and knockdown studies, and cognitive improvement observations in cross studies of HDAC-6 knockdown versus amyloid beta (Aβ)-induced memory deficit models.Taken together, HDAC-6 has a multi-pronged contributory role in neurodegeneration and is a validated druggable target for AD [11].
With regard to OS, in general, excessive radical/nonradical reactive species coupled with diminished brain antioxidant enzyme capacity have been linked to neurodegeneration via a variety of mechanisms (lipid peroxidations, Ca 2+ excitotoxicity, mitochondrial dysfunction, etc.).Additionally, OS also promotes higher HDAC-6 enzyme expression [2,3].OS-induced alterations, therefore, often culminate in synapse dysfunction, which correlates well with cognitive declines in AD patients [4].The aberrantly produced Aβ oligomers also induce reactive oxygen species (ROS) production, leading to neuronal apoptosis [12].Exemplified by glycogen synthase kinase-3 (GSK3) studies, OS can also promote tau hyperphosphorylation, leading to intraneuronal accumulations of neurofibrillary tangles (NFTs) and eventual neuronal death [12].Interestingly, both Aβ and hyperphosphorylated tau beget more OS and the cycle continues.Although antioxidants have performed poorly in clinical trials, there is still discovery space for brain penetrant lipophilic ones, which could potentially minimize lipid peroxidation in neurodegenerative diseases [13].
Since our ultimate goal is to develop single molecules capable of activating σ-1 while inhibiting both HDAC-6 and OS, we designed our compounds to incorporate three key pharmacophoric elements (i.e., two lipophilic groups (Cap or LG1 and LG2) plus a linker) common to both σ-1 agonists [14] and HDAC-6 inhibitors [15] (see Figure 1).We also included the phenolic Trolox moiety in most of our molecules because it has lipophilic, radical scavenging, or antioxidant properties [16] and to possibly add to HDC6 Cap diversity.For the linker group, we utilized the amide functionality because of its Hbonding capability.Essentially, the chemical basis of action for σ-1 ligands involves a minimum of two hydrophobic and one H-bonding interactions compared to at least two hydrophobic interactions plus a ZBG (hydroxamic and non-hydroxamic acids) for HDAC-6 inhibitors [17].The Cap motif acts as a lid and prevents substrates from accessing HDAC's active site, while the ZBG is involved in active site Zn-chelation and H-bonding with the amino acids.Notably, it seems that modifications in the Cap/linker/ZBG functionalities can influence compound selectivity for HDAC isoforms [18].
The rationale for our design is simply that, since σ-1, HDAC-6, and OS (e.g., mitochondria and lipid peroxidation), are independently potentially druggable targets in neurodegenerative pathologies [1,3,19,20], it is plausible that single compounds designed with the trio-target capabilities (i.e., σ-1 activation and HDAC-6 and OS inhibitions) could We also included the phenolic Trolox moiety in most of our molecules because it has lipophilic, radical scavenging, or antioxidant properties [16] and to possibly add to HDC6 Cap diversity.For the linker group, we utilized the amide functionality because of its H-bonding capability.Essentially, the chemical basis of action for σ-1 ligands involves a minimum of two hydrophobic and one H-bonding interactions compared to at least two hydrophobic interactions plus a ZBG (hydroxamic and non-hydroxamic acids) for HDAC-6 inhibitors [17].The Cap motif acts as a lid and prevents substrates from accessing HDAC's active site, while the ZBG is involved in active site Zn-chelation and H-bonding with the amino acids.Notably, it seems that modifications in the Cap/linker/ZBG functionalities can influence compound selectivity for HDAC isoforms [18].
The rationale for our design is simply that, since σ-1, HDAC-6, and OS (e.g., mitochondria and lipid peroxidation), are independently potentially druggable targets in neurodegenerative pathologies [1,3,19,20], it is plausible that single compounds designed with the trio-target capabilities (i.e., σ-1 activation and HDAC-6 and OS inhibitions) could yield novel mechanistic molecules with advantageous neuroprotective properties for testing in AD models.Evidently, there are other ongoing investigations exploring diversely capped HDAC-6 inhibitors for multi-target (e.g., Janus kinase 2 or JAK2 and heat shock protein yield novel mechanistic molecules with advantageous neuroprotective properties for testing in AD models.Evidently, there are other ongoing investigations exploring diversely capped HDAC-6 inhibitors for multi-target (e.g., Janus kinase 2 or JAK2 and heat shock protein 90 or Hsp90) activities as potential synergistic pharmacotherapeutic agents for disparate diseases (e.g., fungal and cancer) [21][22][23].That said, Figure 2 illustrates the 20 phenylacetyl and Trolox amides designed under our proposed pharmacophore hybrid paradigm.

Drug-Likeness
Guided by Lipinski's rule of five, oral drug-like properties depend on the molecule's lipophilicity, flexibility, size, and electronic nature [42,43].We utilized open-source Molsoft (https://www.molsoft.com/servers.html(accessed on 19 July 2023)) computational predictions to identify the above attributes early on in the drug design stages prior to any chemical synthesis [44].Molsoft generated the drug-likeness molecular descriptors (MW, H-bond acceptors or HBAs, H-bond donors or HBDs, LogP, etc.) indicated in Table 1.Representative amidations via HATU or CDI coupling reactions in steps "a" and "b" [40,41], followed by steps "c" and "d" conversions to ZBG functionalities in red.

Drug-Likeness
Guided by Lipinski's rule of five, oral drug-like properties depend on the molecule's lipophilicity, flexibility, size, and electronic nature [42,43].We utilized open-source Molsoft (https://www.molsoft.com/servers.html(accessed on 19 July 2023)) computational predictions to identify the above attributes early on in the drug design stages prior to any chemical synthesis [44].Molsoft generated the drug-likeness molecular descriptors (MW, H-bond acceptors or HBAs, H-bond donors or HBDs, LogP, etc.) indicated in Table 1.
Our target compounds were within the Lipinski drug-like space: MWs < 500), numbers of HBAs (Os + Ns ≤ 10), HBDs (NHs + OHs ≤ 5), and calculated logP values were <5.Low MW small molecules are readily absorbed, diffuse, and are easier to transport across membranes.Additionally, polar surface area (PSA) values for all the molecules were below 90 Å 2 -an indicator of enhanced brain bioavailability [45].Overall, PSA values < 120 Å 2 characterize plausible drug absorption and, therefore, bioavailability and are calculated from surface areas occupied by oxygen, nitrogen, and the attached hydrogen atoms.PSA values are also closely related to the compound's hydrogen bonding capability.Except compounds 5, 16, and 18, the rest of the molecules carried calculated logP values in the range of 2.0-4.5, meaning that they are more capable of crossing biological membranes, including the blood-brain barrier (BBB).Per organic chemistry portal https://www.organic-chemistry.org/prog/peo/(accessed on July 2023), H 2 O solubility (logS) affects drug absorption and distribution, while drug-likeness scores imply the commonness of structural features among target molecules and existing drug molecules.Our target compounds were within the water H 2 O solubility range of −2 to −4 for most marketed drugs.Also, most of our compounds (except 1, 5, 16, and 18) exhibited positive scores-an indication that they contained most of the usual functionalities found in clinical drugs.[47].The two-step assay protocol involved 1) incubating the purified HDAC enzyme with a fluorogenic substrate possessing an acetylated lysine side chain and 2) treating the above incubated substrate with a developer to produce a fluorophore.Notably, deacetylation of the substrate sensitizes it for the second step.All compounds were dissolved in DMSO and tested in at least 10-dose IC50 mode with threefold serial dilutions (Table 3)."(R)-Trichostatin A or (R)-TSA", a potent (e.g., IC50s between 6 and 38 nM) in vivo/in vitro inhibitor of human HDACs, was used as a comparator [48,49].Percent enzyme activities (relative to DMSO controls) and IC50 values were calculated using GraphPad Prism 4 program (Figure 3).[47].The two-step assay protocol involved 1) incubating the purified HDAC enzyme with a fluorogenic substrate possessing an acetylated lysine side chain and 2) treating the above incubated substrate with a developer to produce a fluorophore.Notably, deacetylation of the substrate sensitizes it for the second step.All compounds were dissolved in DMSO and tested in at least 10-dose IC50 mode with threefold serial dilutions (Table 3)."(R)-Trichostatin A or (R)-TSA", a potent (e.g., IC50s between 6 and 38 nM) in vivo/in vitro inhibitor of human HDACs, was used as a comparator [48,49].Percent enzyme activities (relative to DMSO controls) and IC50 values were calculated using GraphPad Prism 4 program (Figure 3).

ORAC Assay
Compound antioxidant capacities were determined using the Oxygen Radical Activity Capacity (ORAC) Assay Kit (catalog #ab233473, Abcam, Cambridge, MA, USA) according to the manufacturer's protocol.Briefly, the Trolox standard (0.2 mM, 50:50 v/v, water: acetone) and each test compound (0.2 mM, 50:50 v/v, water: acetone) were tested at final concentrations ranging from 2.5 µM to 50 µM in the assay.Fluorescein (150 µL) probe solution and 25 µL of Trolox or each test compound were mixed in each well of a black 96-well microplate and incubated at 37 • C for 30 min.Wells which did not contain any antioxidant standard (Trolox) or test compounds served as blanks.The radical initiator (2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH) solution (80 mg/mL, 25 µL) was then added to each well to start the reaction.Immediately, the microplate was placed in a fluorescence microplate reader (BioTek Synergy HTX) and fluorescence was measured at respective wavelengths of 480 nm (excitation) and 520 nm (emission) at 37 • C. Relative fluorescence was measured in increments of 1 min for 60 min.Fluorescence values of Trolox and test compounds for each well were then plotted against time.Individual net areas under the curve (AUC, determined by subtracting the AUC of the blank) were then plotted against different concentrations of Trolox and test compounds to generate calibration curves.Trolox equivalents (TE) for the test compounds were obtained by directly dividing the slopes of each sample by the slope of the Trolox from its standard curve (Table 4) [50].All compounds were initially screened for antioxidant activity and the six most active ones (i.e., 3, 4, 8, 12, 18, and 20) were run in triplicates to obtain error bars-this data is plotted in the Supplementary Materials section.Curiously, unlike the phenolic Trolox derivatives (i.e., 3, 4, 8, 12, and 20), compound 18 s antioxidant activity can be attributed to the -NH-OH moiety [51].

Compound 8 Modelling
Modelling was undertaken to determine and contrast the docking poses using overlays (Figure 5A,B), binding energy scores or affinities, and two-dimensional amino acid (AA) bonding interactions (Figure 6A-D

Compound 8 Modelling
Modelling was undertaken to determine and contrast the docking poses using overlays (Figure 5A,B), binding energy scores or affinities, and two-dimensional amino acid (AA) bonding interactions (Figure 6A-D) of our promising lead compound 8 versus native ligands Trichostatin A (TSA) for HDAC-6 and PD144418 for σ1R.The applicable docking protocol details are described elsewhere [52].Briefly, energy minimized conformers were converted into Protein Data Bank, Partial Charge (Q), and Atom Type (T) or pdbqt format.Crystal structures of HDAC-6 complexed with Trichostatin A and σ1R complexed with PD144418 were obtained from the Protein Data Bank database [https://www.rcsb.org/(accessed on 5 October 2023), PDB, ID: HDAC-6-5EDU, σ1R-5HK1] and imported into Chimera 1.16 to visualize the binding region of the complexes and identify key amino acid (AA) residues involved.The most stable HDAC-6/5EDU bound conformers of 8 exhibited free-binding energy or affinity scores (in the range −9.1 to −7.2 kcal/mol) comparable to the reference ligand TSA (−8.1 kcal/mol).Notable H-bonding interactions were between the -NH-OH group and GLY 619 or TYR 782.Other interactions included van der Waals forces: πstacked (PHE 680, PHE 620) for 8, π-sigma (SER 568, PHE 680, PHE 620) for TSA, plus conventional dispersion forces displayed in both ligands (i.e., via LEU 749, HIS 651, HIS   The most stable HDAC-6/5EDU bound conformers of 8 exhibited free-binding energy or affinity scores (in the range −9.1 to −7.2 kcal/mol) comparable to the reference ligand TSA (−8.1 kcal/mol).Notable H-bonding interactions were between the -NH-OH group and GLY 619 or TYR 782.Other interactions included van der Waals forces: πstacked (PHE 680, PHE 620) for 8, π-sigma (SER 568, PHE 680, PHE 620) for TSA, plus conventional dispersion forces displayed in both ligands (i.e., via LEU 749, HIS 651, HIS The most stable HDAC-6/5EDU bound conformers of 8 exhibited free-binding energy or affinity scores (in the range −9.1 to −7.2 kcal/mol) comparable to the reference ligand TSA (−8.1 kcal/mol).Notable H-bonding interactions were between the -NH-OH group and GLY 619 or TYR 782.Other interactions included van der Waals forces: π-stacked (PHE 680, PHE 620) for 8, π-sigma (SER 568, PHE 680, PHE 620) for TSA, plus conventional dispersion forces displayed in both ligands (i.e., via LEU 749, HIS 651, HIS 611, HIS 610, and HIS 500) added to the stability of lipophilic cap areas.On the other hand, the best docked σ1R/5HK1 bound conformers of 8 scored in the range of −10.9 to −9.3 kcal/mol.These scores were also comparable to the native co-crystallized ligand PD144418 docked at 5HK1 (−10.2 kcal/mol).Compound 8 displayed a polar H-bond interaction between its -NH-OH and ASP119, while PD144418 did not display any H-bonding.The most notable interactions shared by both docked ligands were π-anion interactions (GLU 165 and ASP 119), π-alkyl interactions (ALA 178, MET 86, and LEU 98), π-π T-shaped interactions with PHE 10, and π-sigma interactions (HIS 147 and TYR 96), which contributed to the stability of the cap and lipophilic regions of the ligands.

Materials and Methods
Reactants and solvents were sourced from Thermo Fisher Scientific and Acros Organics, (Pittsburgh, PA, USA), Millipore Sigma-Aldrich (St. Louis, MO, USA), eMolecules (San Diego, CA, USA) and used as received.All reactions were performed according to literature precedence.Melting points, taken on the Mel-temp capillary apparatus, are reported uncorrected.Teledyne Combiflash Rf flash chromatography fitted with Redisep Rf silica gel cartridges was used for compound purifications.Analytical TLC plates from EM Science (silica Gel 60 F 254 ) were used. 1 H and 13 C spectra were recorded using CDCl 3 or DMSO-d 6 as a solvent on a Bruker 300 MHz spectrometer at ambient probe temperature, unless otherwise indicated. 1H and 13 C chemical shifts are reported versus SiMe 4 and were determined by reference to the residual 1 H and 13 C solvent peaks.Coupling constants (J) are reported in hertz (Hz).Characterization data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constants, number of protons, and mass-to-charge ratio.Purities and masses of the 20 compounds were determined by analytical reversed-phase high-performance liquid chromatography/mass spectrometer (HPLC/MS) tandem on a Dionex Ultimate 3000 HPLC system.The HPLC method utilized Nova-Pak C18 Column, 60 Å, 4 µm (4.6 mm × 150 mm), at ambient temperature and a flow rate of 1.0 mL/min., CH 3 CN/H 2 O eluent (containing 0.1% acetic acid); gradient, 5% CH 3 CN to 100% CH 3 CN; 8 min.; and UV detection at 254, 260, and 280 nm.nm.Mass was obtained in positive ion mode using heated electrospray ionization (ISQ-HESI) source.MS conditions: capillary voltage, 3.0000 V; drying gas flow, 0.2 mL/min; vaporizer gas temperature, 350 • C; and gas pressure, 28.8 psig.MS data were acquired with Chromeleon 7.

Chemistry
Procedure A-Acylation amidation using modified organic "Schotten-Baumann" conditions [36].Phenyl acetyl chloride (1.0 equiv.) was added dropwise to a solution of the appropriate amine (1.0 equiv.)and N, N-diisopropylethylamine (DIPEA, 3 equivs.)in 2:1 dichloromethane (DCM)/methanol (MeOH) co-solvent at 0 • C. The ensuing mixture was stirred from 0 • C to room temperature (RT) overnight, under N 2 .The reaction was quenched with H 2 O and extracted with DCM.The pooled organic phase was sequentially washed with Na 2 CO 3 and brine, dried over Na 2 SO 4 , concentrated by rotary evaporator, and purified by flash chromatography using either hexane/ethyl acetate (7:3) or DCM/MeOH (9:1).Solvent removal from product fractions (per TLC) or recrystallizations from DCM afforded the target compounds.Yields are indicated in the experimental write-up of each product.
Procedure B-HATU amidation.A mixture of Trolox (1.0 eqiv.), the appropriate amine (1.5 eq), and HATU (1.5 equivs.) in dimethylformamide (DMF) was stirred at room temperature (RT) for 10 min under N 2 .DIPEA (2.5 equivs.)was added to the reaction mixture, followed by overnight (18-24 h) stirring.The reaction was diluted with DCM, washed with excess H 2 O, and the combined organic phase was washed with brine then dried over Na 2 SO 4 .Product purification was achieved by flash chromatography using hexane/ethyl acetate (7:3) or DCM/MeOH (9:1) as eluents.Solvent removal/recrystallization of product fractions afforded the desired compounds in the yields indicated in individual experimental write-ups.Reaction solvent amounts are included in each experimental write-up.
Procedure C-CDI amidation.After 1 h of stirring 1 -carbonyldiimidazole (CDI) (1.5 equivs.)and Trolox (1.0 equiv.) in tetrahydrofuran (THF) (10 mL), the appropriate amine (1.5 equivs.)was added.The resulting reaction mixture was further stirred, then stirred overnight (24 h) at RT under N 2 .THF was removed under vacuo, H 2 O was added, and organics were extracted with DCM.The combined organic phase was washed with Na 2 CO 3 and brine, then dried with Na 2 SO 4 .After solvent removal, the crude product was purified via flash chromatography (Combi-Flash Rf) using hexane/ethyl acetate (7:3) or DCM/MeOH (9:1) eluent options.Collection of the relevant fractions yielded the product amounts illustrated in individual experimental write-ups.
Procedure D-Hydrazide synthesis.Excess hydrazine hydrate (50 equivs.)was added in a drop-wise manner to the appropriate methyl ester intermediate (1 equiv.) in absolute ethanol (EtOH) and the reaction mixture was refluxed overnight (24 h).The precipitate which formed was filtered and purified by successively rinsing the solids with cold (0 • C) H 2 O. Reaction solvents and product yields are reported in respective experimental write-ups.
Procedure E-Hydroxamic acid synthesis.Hydroxylamine hydrochloride (15 equivs.)was freebased by stirring it with NaOH (30 equivs.) in MeOH for 10 min and filtered.The methyl ester intermediate (1 equiv.) was added to the hydroxylamine freebase filtrate and the mixture stirred at RT overnight (18 h).The reaction mixture was concentrated by rotary evaporator, dissolved in H 2 O (10 mL), and the solution was acidified to 4 pH with HCl (5N).The crude hydroxamic acid precipitate which formed was filtered and rinsed with ice-cold H 2 O to afford the product.Reaction solvents and product yields are reported in respective experimental write-ups.
Procedure F-Hydrolysis.Excess NaOH (8 equivs.)was added to the appropriate methyl ester intermediate (1 equiv.) in absolute EtOH/THF (10 mL, 1:1) co-solvent.The reaction mixture was refluxed for 4 h and concentrated under vacuo.Water (10 mL) was added and the solution was acidified to 4 pH with HCl (5N).The solid COOH precipitate which resulted was filtered and purified by rinsing with ice-cold H 2 O. Reaction solvents and product yields are reported in experimental write-ups (e.g., compound 17).
Author Contributions: All authors contributed to the successful completion of this project.D.S. and R.F. synthesized, performed NMR/HPLC-MS analysis.D.S. conceived, wrote and edited the manuscript.R.F. wrote the experimental section including the NMR supplementary data.ORAC experimental data collection and analysis was done by R.F. and S.I.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.Our proposed hybrid model incorporating pharmacophoric elements of σ-1 (i.e., LG1, Hbonding linker, and LG2) and HDAC (i.e., Cap or LG1, linker for molecule insertion into the active site gorge, and an LG2 with zinc binding group or ZBG) ligands.Antioxidant adornments could be part of either LG1 or LG2 motifs.

Figure 1 .
Figure 1.Our proposed hybrid model incorporating pharmacophoric elements of σ-1 (i.e., LG 1 , Hbonding linker, and LG 2 ) and HDAC (i.e., Cap or LG 1 , linker for molecule insertion into the active site gorge, and an LG 2 with zinc binding group or ZBG) ligands.Antioxidant adornments could be part of either LG 1 or LG 2 motifs.

Figure 2 .
Figure 2. The 20 synthesized target small molecules possessing two lipophilic/hydrophobic regions and a common amide linker.Potential or promising leads are in red.

Figure 2 .
Figure 2. The 20 synthesized target small molecules possessing two lipophilic/hydrophobic regions and a common amide linker.Potential or promising leads are in red.

Figure 4 .
Figure 4. TEs for six compounds versus Trolox standard determined by ORAC assay after 60 min.Data are presented as means ± standard deviation (SD), n = 3.

Figure 4 .
Figure 4. TEs for six compounds versus Trolox standard determined by ORAC assay after 60 min.Data are presented as means ± standard deviation (SD), n = 3.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 8 of 17 Crystal structures of HDAC-6 complexed with Trichostatin A and σ1R complexed with PD144418 were obtained from the Protein Data Bank database [https://www.rcsb.org/(accessed on 5 October 2023), PDB, ID: HDAC-6-5EDU, σ1R-5HK1] and imported into Chimera 1.16 to visualize the binding region of the complexes and identify key amino acid (AA) residues involved.

Table 1 .
Molsoft-predicted drug-likeness pharmacokinetic properties for the synthesized compounds.

Table 1 .
Molsoft-predicted drug-likeness pharmacokinetic properties for the synthesized compounds.

Table 4 .
Slopes and TEs for the six compounds, plus Trolox.These data were transfigured into Figure4.each well to start the reaction.Immediately, the microplate was placed in a fluorescence microplate reader (BioTek Synergy HTX) and fluorescence was measured at respective wavelengths of 480 nm (excitation) and 520 nm (emission) at 37 °C.Relative fluorescence was measured in increments of 1 min for 60 min.Fluorescence values of Trolox and test compounds for each well were then plotted against time.Individual net areas under the curve (AUC, determined by subtracting the AUC of the blank) were then plotted against different concentrations of Trolox and test compounds to generate calibration curves.Trolox equivalents (TE) for the test compounds were obtained by directly dividing the slopes of each sample by the slope of the Trolox from its standard curve (

Table 4 .
Slopes and TEs for the six compounds, plus Trolox.These data were transfigured into Figure 4.