Histone acetylation is a type of epigenetic change strictly controlled by histone deacetylases (HDACs) and histone acetyltransferase (HAT). HDAC enzymes are responsible for removing the acetyl group from the histone lysine residues [1]. The high population of the free lysine \(\epsilon\)-amino group allows the tight interaction between the histone and DNA’s negative charge. In addition, hypoacetylation impairs angiogenesis, migration, invasion, and cell adhesion, eventually initiating and advancing cancers [2,3]. There are eighteen distinct HDAC isoforms in all, grouped into four groups. Class II (HDAC4-7, 9, and 10), class III (SIRTs1-7), class IV (HDAC11), and class I (HDAC1, 2, 3, and 8)[4]. Zinc ion is necessary for HDACs 1 through 11 to function as catalytic enzymes. The suppression of the HDAC enzyme requires zinc chelation. It is widely known that overexpression of HDACs occurs in several illnesses, including cancer, HIV, neurological diseases, and cardiovascular disorders[5].

Four HDAC inhibitors have been used to treat cancer [6, 7]. HDAC inhibitor structures are often identified by the presence of a zinc-binding group (ZBG), a cap, and a connecting linker. The primary mechanism by which HDAC inhibitors impede their function is the binding of zinc-binding glycosides (ZBGs) to the zinc ion and adjacent residues, as the cap and linker maximize the HDAC inhibitors’ efficacy and selectivity. Developing new HDAC inhibitors with optimum structural features is a high need. In continuous with our work [8, 9, 10, 11], new molecules with possible HDAC inhibition activity were synthesized and biologically evaluated.

Materials and Method

Solvents and chemicals were supplied as the source supplied with no further purification. The \(Alugram^\circledR\) Xtra SIL G/UV254 (Macherey-Nagel, Germany) 0.2 mm pre-coated TLC-sheets were used, and the visualization was done under a 254 nm UV lamp-TLC to track the reaction’s course. The Stuart SMP3 melting point apparatus (UK) was used to determine the melting points in open capillary tubes. Specac\(^\circledR\) Quest ATR-diamond type (UK) and Shimadzu IRAffinity-1 Spectrometer (Shimadzu, Japan) were used for Fourier Transform Infrared Spectroscopy (FT-IR). The Bruker 500 MHz-Avance III spectrometer (USA) was used to conduct nuclear magnetic resonance (NMR) spectroscopy.

Molecular Docking

A docking study was conducted utilizing the licensed Glide module impeded in the Maestro program, part of Schrodinger’s modeling software version 13.0135. Vorinostat is the designated reference chemical for modeling purposes. The zinc-binding group fragments were produced via the ligand designer module inside the program, [12] to assess the binding affinity and inhibitor activity of compounds (IVa-d) into the HDAC2, HDAC6, and HDAC8.

The Ligprep module was utilized to build the chemical structure for the proposed compounds. The crystal structure for targets, HDAC2 (4LXZ) [13], HDAC8 (1W22) [14], HDAC6 (2VQO) [15] were downloaded from protein data bank[16] and prepared using protein preparation wizard from Maestro including preprocessing of the protein to assign bond order, replacing missed hydrogen, adding terminal oxygen to the chain, deleting water beyond \(3A^\circ\), forming bond fewer than three bonds with non-water molecules, and generating ligand [17]. They enhanced hydrogen bond assignments by using the default configuration and refining them via reduction. A single chain without any amino acid breaks inside the active site was chosen for dimer or trimer proteins. The active site for docking was determined by generating a grid based on the co-crystallized ligand acquired from PDB. The default settings were used, with the grid size limited to \(15A^\circ\) * \(15A^\circ*15A^\circ\) [18, 19]. The ligands derived via ligprep were subjected to high precision (XP) docking analysis against HDAC2,6,8. The docking findings were assessed by evaluating the ligand’s fitness to the active site, docking score, types and number of bonds formed, Van der Waals and Columbus forces energy, Gscore, and other relevant variables [18, 19].

ADMET Study

The developed compounds were subjected to pharmacokinetic property prediction using the default configuration of QIKPROP, a licensed software from the Schrodinger suite. The chemical used as a model is a vorinostat, in which the hydroxamate group is substituted with an amide or sulfonamide moieties that serve as a zinc-binding group (ZBG).

Organic Synthesis

Figure 1: The Organic Synthesis for final Compounds

General Method for Amide Synthesis (IIIa&b)

To a round bottom flask added 3-(4-bromophenyl) propanoic acid (Ia) or 3-(3-bromophenyl) propanoic acid (Ib) (5 mmol, 1 equiv), bromoethylamine hydrobromide (II) (1.23 g, 5 mmol, 1.2 equiv), EDCI (1.146 g, 5 mmol, 1.2 equiv) HOBT (0.07 g, 5 mmol, 0.1 equiv), DMAP (0.732 g, 5 mmol, 1.2 equiv), DIPEA (0.39 g, 3 mmol, 3 equiv) and dry DCM (7 mL) were stirred under argon for 36 hours The progress of reaction was monitored by TLC (EtOAc: Hex, 3:1). As the reaction is completed the mixture was purified by column chromatography using (EtOAc /Hex as eluent) to afford (cpd IIIa and IIId) as yellow crystals in yield 70-80%. IIIa, IR (\(v\), \(cm^{-1}\)): 3286 (N-H ), 3059-2881 (C-H str.), 1666 (C=O str. amide), 1597-1566 (C=C str. aromatic ring), 779 (C-Br). 1H NMR (500 MHz, CDCl3) \(\delta\) 8.0 (s, 1H), 7.35 - 6.7 (m, 4H), 3.6 (m, 4H), 2.7 (d, J = 42.6 Hz, 2H), 2.4 (s, J = 37.7 Hz, 2H). IIIb, IR (\(v\), cm-1): 3298 (N-H amide), 3062 -2854 (C-H str.), 1651 (C=O str. amide), 1597 (C=C str. aromatic ring), 744 (C-Br). 1H NMR (500 MHz, CDCl3) \(\delta\) 8.1 (s, 1H), 7.8 - 7.4 (m, 4H), 3.7 (d, J = 42.6 Hz, 2H), 3.4 (d, J = 37.7 Hz, 2H), 2.6 (d, J = 42.6 Hz, 2H), 2.5 (d, J = 37.7 Hz, 2H).

General Method for Synthesis of Compounds IVa-IVd

To a round bottom flask added (\(p\)-amino acetanilide) (405 mg, 2 mmol, 1 equiv) or (N-(4-aminophenyl)(methane sulfonamide) (560 mg, 2 mmol, 1 equiv), TEA (0.6 mL, 2 equiv) and dry DMF 3 mL. The mixture was stirred under argon and heat at \(40^\circ\)C with stirring for 1 h (cpd IIIc&d) (2 mmol, 1 equiv) were added separately gradually and stirred at \(80^\circ\)C for 24 hours. The reaction was monitored by TLC (EtOAc: Hex, 3:1). As the reaction was completed, the solvent removed by dry air and the product was purified using Combiflash eluted with EtOAc/ Hex (20-100%) to produce final compound (VIa-VId) as yellow-brown crystals in 35-45% yield. Iva, m.p. 184-189; IR (\(v\), \(cm^{-1}\)): 3294,3232 (N-H str.), 3010-2931 (C-H str.), 1658 (C=O str. amide), 1600 (C=C str. aromatic ring), 1527,1315 (\(SO_{2}\) str.), 779 (C-Br). 1H NMR (500 MHz, \(CDCl_{3}\)) \(\delta\) 8.2 - 7.9 (s, 2H), 7.4 - 7.1 (m, 8H), 3.7 - 3.6 (s, 4H), 3.4 (d, J = 42.6 Hz, 2H), 3.1 (d, J = 42.6 Hz, 2H), 1.5 (s, 3H). IVb, m.p. 176 - 181; IR (\(v\), cm-1): 3300-3200 (N-H str.), 3059-2958 (C-H str.), 1651 (C=O str. amide), 1597 (C=C aromatic ring), 1527,1315 (SO2 str.), 748 (C-Br). ). 1H NMR (500 MHz, \(CDCl_{3}\)) \(\delta\) 8.2 - 8.1 (s, 2H), 7.4 - 7.1 (m, 8H), 3.6 (m, 4H), 3.2 (d, J = 42.6 Hz, 2H), 2.7 (d, J = 37.9Hz, 2H), 2.4 (d, J = 42.6 Hz, 2H), 1.7 (s, 3H). IVc, IR (\(v\), \(cm^{-1}\)): 3300-3200 (N-H str.), 3078-2854 (C-H str.), 1678,1647 (C=O str. amides), 1600 (C=C aromatic ring), 759 (C-Br). 1H NMR (500 MHz, \(CDCl_{3}\)) \(\delta\) 8.2 - 7.8 (s, 3H), 7.6 - 7.1 (m, 8H), 3.9 (d, J = 37.9Hz, 2H), 3.2 (d, J = 42.6 Hz, 2H), 2.8 (d, J = 37.9Hz, 2H), 2.1 (d, J = 42.6 Hz, 2H), 1.65 (s, 3H).

Cancer Cell Cytotoxicity Study

This work used an MTT study to evaluate the cell growth inhibition [20]. vorinostat, Iva, and IVb were incubated with colon cancer cells (174-TB16). The stock for all compounds was 0.1 mg/1 mL, with serial dilution at 50% for each (100, 50, 25, 12.5, 6.25, 03.13 \(\mu\)g/mL). Add 2 \(\mu\)L of each dilution into 198 \(\mu\)L of the cancer cell line and incubate for 24 hours at \(37^\circ\)C. After the drug exposure period is complete, prepare a solution of MTT reagent and add it to each well; incubate the plate for 4 hours to allow MTT to be converted to formazan. After that, each well was filled with 200 \(\mu\)L of the resulting solution. The plate was incubated at \(37^\circ\)C for 4 hours until purple intracellular formazan crystals were visible under an inverted microscope. After removing the supernatant, 100 \(\mu\)L of DMSO was added to each well to dissolve the resultant formazan crystals. The plate was incubated for 30 minutes at room temperature until the cells were lysed and the purple crystals dissolved. The percentage of cell viability or proliferation was calculated by dividing the absorbance readings of test samples by those of the control samples and multiplying by 100 to determine the IC₅₀ value.

Molecular Docking

To evaluate the in silico potency and selectivity for Iva and , a molecular docking study was carried out on variant HDAC isoforms of HDAC2, HDAC6, and HDAC8. The docking score for the final compounds on HDAC isoforms was significantly higher than the FDA-approved drug of vorinostat (Table 1). The representative virtual interaction of Iva with HDAC6 shows that the sulfonamide moiety forms a monodentate interaction with zinc ion and forms two hydrogen bonds with Gly167 and His158 residues inside the active site. Additionally, the linker aromatic group forms \(\pi-\pi\) stacking with Phe227. Finally, the \(m\)-bromophenyl cap group is probing outside the active site to stabilize the interaction (Figure 2).

Figure 2: The 2D Interaction of Iva with HDAC6

ADME study

Prior to synthesis or preliminary studies, it is crucial to engage in virtual prediction of medicinal characteristics. Compounds Iva and IVb showed acceptable \(in silico\) pharmacokinetic properties. In addition, molecules having decent calculated oral absorptivity and no violation for drug-likeness rules (Table 2).

Rtvfg: Range of reactive functional group counts, from zero to two, a lower value is preferable since it indicates that the functional group is less stable and hence more likely to break down to other groups or have a hazardous impact.

Rule of Five: Number of times Lipinski’s rule of five was broken. Donor HB 5, Receiver HB 10, and Molecular Weight (MW) 500 are the guidelines. Substances meeting these criteria are classified as potential pharmaceuticals.

Rule of Three: Jorgensen’s rule of three breakdowns in frequency. QPlogS > -5.7, QP PCaco > 22 nm/s, and Primary Metabolites 7 are the three criteria to follow. Orally, bioavailable compounds break these criteria less often, if at all.

Chemistry

The synthesis of final compounds was commenced with the amidation reaction between bromoethylamine hydrobromide (II) with various acids of 3-(4-bromophenyl)propanoic acid (Ia) or (3-(3-bromophenyl) propanoic acid (Ib) to produce amides (IIIa & IIIb). Using TEA in dry DMF under argon with heating to 800C, the p-amino acetanilide or (N-(4-amino phenyl) methane sulfonamide added to compounds (IIIa & IIIb) to afford final compounds (IVa and IVb)) (Figure 1).

The preliminary MTT assay against colon cancer cell line (LS-174T) indicated a promising preliminary cytotoxic activity for the synthesized molecules. Sulfonamides Iva and IVb have a sub-micromolar activity which is comparable to the FDA approved drug vorinostat. With half maximal inhibitory concentration (\(IC_{50}\)) of 0.37 \(\mu\)M and 0.44 \(\mu\)M, respectively, while vorinostat has an \(IC_{50}\) of 0.7 \(\mu\)M (Figure 3).

Figure 3: The Dose-Response Inhibition Assay of (A) Vorinostat, (B) Iva, (C) Ivb Against Colon

The instillation of sulfonamide moiety new ZBG designed new HDAC inhibitors. The molecular docking studies showed an acceptable virtual interaction of compounds Iva and IVb with HDAC enzymes. Final compounds were synthesized utilizing conventional organic synthesis methods. The in silico ADME studies revealed an acceptable drug-likeness. The preliminary antiproliferative study on cancer cells of LS174T indicated that compounds IVa and IVb have promising colon cancer cell growth inhibition activity with a sub-micromolar IC50 and the activity is comparable to vorinostat.

The work is partially funded by the College of Pharmacy, University of Baghdad, Iraq.

The authors declare no conflicts of interest.

All authors contributed equally to this paper. They have all read and approved the final version.

Informed consent was obtained from all participates in the study as needed.

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