Design, Synthesis and Cytotoxicity Study of Primary Amides as Histone Deacetylase Inhibitors

Primary amide derivatives as histone deacetylase inhibitors (HDACIs) are very rare. This paper describes the synthesis of the primary amide derivatives adipic monoanilide amide and pimelic monoanilide amide (compounds 6 and 7) that have the requirements to be histone deacetylase inhibitors of the zinc-binding type. Both of them exhibited good cytotoxicity against the tested cancer cell lines with much lower cytotoxicity against normal cell line.


Introduction
Histone deacetylases (HDACs) are a group of enzymes responsible for the deacetylation of the ε-amino groups of lysine residues at the N-termini of histones (1) , the core proteins that serve to compact DNA into nucleosomes which comprise chromatins (2) . The deacetylation process encourages the compaction of nucleosomes and repression of gene transcription (3) . Many reports demonstrate that HDACs are overexpressed in several types of cancer (1,4) . Therefore , they are considered as valuable targets for cancer treatment (5) .
Eighteen isoforms of HDACs have been identified in mammalian tissues. They fall into four major classes (I-IV). Class I includes HDAC1, 2, 3, and 8 while class II includes isoforms 4-7, 9 and 10.
Isoform 11 represents class IV. These three classes have a zinc-dependent catalytic site. The rest seven isoforms depend on the cofactor nicotine adenine dinucleotide rather than zinc for their catalytic activity and comprise class III (6) .
The zinc-dependent classes are highly investigated and subjected to inhibition studies as an approach for cancer treatment (7) . A classical and popular example is HDAC2 since its crystallographic structure is well resolved (8) . Previous studies on the binding site revealed that it is shaped as an internal tubular (tunnel) cavity having a length of 11 Å. At the bottom of this cavity lies a zinc ion followed by a 14 Å foot-like pocket (9) (Figure 1). Lysine residues of histone fit into the tunnel so that they could be deacetylated (3) . Accordingly, the design of inhibitors for zinc-dependent HDACs should have a zinc binding group (ZBG) to bind to the zinc ion, a linker of 4-6 carbon atoms length to fit into the cavity, and a hydrophobic cap to recognize and interact with the outer surface (1) . These are best illustrated by the classical inhibitor suberoylanilide hydroxamic acid 1 (SAHA, vorinostat) (10) .Other known synthetic inhibitors with ZBG other than hydroxamic acid derivatives include entinostat 2 (MS-275), a benzamide, and valproic acid 3 (5) (Figure 2). Among other rare synthetic ZBG are the secondary amide derivative 4 (11,12) and the only primary amide 5 mentioned in the literature that showed an IC50 against HDAC1 in a nanomolar concentration (13) .

Figure2. Examples of HDAIs with different ZBGs
Here, we wish to describe the synthesis of the small and simple primary amide molecules 6 and 7 that fulfill the three requirements for zinc-dependent HDAC inhibitors.
(6) n= 2 (7) n= 3 Besides, we'll mention the results of the in vitro cytotoxicity assay for these compounds against HRT-18 cell line (human colon adenocarcinoma) and HC-04 cell line (mouse hepatic carcinoma).

Materials and Methods
Reagents and solvents were used for chemical synthesis as obtained from the supplier (Sigma-Aldrich, Fluka, Romil, GCC Diagnostics, ReagentWorld, and Thomas-Baker). Melting points were measured using the Stuart SMP3 melting point apparatus (UK) and are uncorrected. Thin-layer chromatography was achieved using 0.2 mm precoated TLC-sheets Alugram ® Xtra SIL G/UV254 (Macherey-Nagel, Germany) and the visualization was under a 254 nm UV lamp. FT-IR spectroscopy was done using Shimadzu IRAffinity-1 Spectrometer (Shimadzu, Japan) and Specac ® Quest ATR-diamond type (UK) at the University of Baghdad-College of Pharmacy. 1 H-NMR and 13 C-NMR analysis was performed at 400 MHz and 100 MHz respectively (d6 -DMSO as the solvent) using The Bruker Avance III, 400 MHz spectrometer (USA) at Sophisticated Test and Instrumentation Centre (Cochin University of Science and Technology-India), with the chemical shifts (δ) expressed in parts per million.
Figures illustrating the binding site of HDACs and the binding of the ligand were captured using UCSF Chimera (14) and the PDB (www.rcsb.org) (15) files with IDs 4LXZ (8) and 3MAX (16) .

General synthesis of monosodium adipic monoanilide and monosodium pimelic monoanilide (6d and 7d)
The obtained mixture of compounds 6b and 6c, or 7b and 7c was dissolved in 10 mL of dry DMF in a round bottom flask that was equipped with calcium chloride tube and cooled to 0 °C . Then 3 mL of aniline (32.88 mmol) were added gradually with continuous stirring and cooling. The mixture was then kept stirred at room temperature for 24 hours. The mixture was then acidified with 5 N hydrochloric acid, diluted to about 300 mL with cold water, and the precipitate formed was then filtered. The precipitate was washed with water (3×50 mL). Then it was suspended in no more than 50 mL of icecooled water and the pH of this mixture was raised to 7 by a drop wise addition of 0.5 N sodium hydroxide solution at 0 °C . The mixture was stirred for 30 minutes at 0 °C while maintaining this pH by addition of 0.5 N sodium hydroxide solution as needed. Then the mixture was filtered and the filtrate was evaporated to dryness. Compound 6d or 7d (4 mmol) was suspended in 15 mL dry dichloromethane in a round bottom flask that was equipped with calcium chloride tube and cooled in an ice bath. Pyridine (4 mmol, 0.32 mL) was added followed by a drop wise addition of 0.34 mL of thionyl chloride (4.8 mmol) with continuous stirring and cooling for 10 minutes. The mixture was then allowed to warm to room temperature with continuous stirring for 30 minutes. Then the precipitate formed was collected using Büchner funnel. The precipitate was washed with dry dichloromethane (3×10 mL) during filtration. The white solid mass obtained (in both cases of 6e and 7e) was used as such in the next step.

General synthesis of adipic monoanilide amide and pimelic monoanilide amide (6 and 7)
The precipitate obtained from the previous step (6e or 7e) was suspended in 10 mL of anhydrous cold acetone and added gradually to 10 mL of concentrated aqueous ammonium hydroxide solution that was cooled in an ice bath. The mixture was stirred for 30 minutes while maintained in the ice bath, then extracted three times with 5 mLportions dichloromethane. The organic phase was evaporated to dryness under reduced pressure.

Cytotoxicity assay
MTT cell viability assay was done using 96-well plates. The chosen cell lines were seeded at 1 × 10 4 cells/well. After either 24 hours or a confluent monolayer had been obtained, the cells were treated with compounds 6 or 7 at different concentration (6.25, 12.5, 25, 50, 100 µM). After a 72 hours-treatment period, the cell viability was measured by removing the medium, adding 28 µL of 2 mg/mL solution of MTT stain and incubating the cells for 2.5 hours at 37 °C. Then the MTT solution was removed and the remaining crystals in the wells were dissolved in 130 µL of dimethyl sulfoxide followed by 37 °C incubation for 15 minutes with shaking (18)] . The absorbency was determined on a microplate reader at 492 nm; the assay was performed in triplicate.
The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated according to the following equation: where A is the optical density of the control and B is the optical density of the sample. The morphology of cells was visualized by the aid of inverted microscope. 200 µL of cell suspensions were seeded in 96-well micro-titration plates at density 1x10 4 cells/ mL and incubated for 48 hours at 37 °C. After removing the medium, compound 6 or 7 was added at the (IC50). Then, after the exposure time, 50 µL of crystal violet were used to stain the plates which were incubated at 37 °C for 15 min; gentle washing of the stain was done with water until no more dye remained. A 40x magnification of inverted microscope was selected to examine the cells; they were photographed with a digital camera.

Statistical analysis
Unpaired t-test (using GraphPad Prism 6) was applied for the statistical analysis of the collected data. The values were presented as the mean ± SEM of triplicate measurements.

Chemical synthesis
Synthesis of the target compounds (6 and 7) was started from adipic acid (6a) and pimelic acid (7a) respectively (Scheme 1). Heating these dicarboxylic acids in acetic anhydride for 1 hour would result in monomeric cyclic anhydrides (6b and 7b). However, these anhydrides are extremely unstable and some of the synthesized amount is converted into polymeric anhydrides (6c and 7c) rapidly during evaporation of the solvent (11,19) . The anhydride mixture (6b and 6c, or 7b and 7c) was used as such in the next step which is a modified procedure from previously published works (20,21) . In this step, a slight excess of aniline (1.2 eq. relative to 6a and 7a) was added gradually to an ice-cooled solution of the anhydride mixture (in dry DMF) and, then, stirred at room temperature for 24 hours. The sodium salts (6d and 7d) were obtained by treatment of the precipitate, resulted after cold acidification of the reaction mixture and filtration, with gradually increasing amounts of ice-cooled NaOH solution (0.5 N) so that the pH was not raised above 7. After filtration and evaporation to dryness, the off-white precipitate (6d and 7d) showed the characteristic FT-IR amide peaks (respectively) at 3332 cm -1 and 3290 cm -1 (aromatic NH) and at 1666 cm -1 and 1654 cm -1 (amide carbonyl). Furthermore, the carboxylate peaks were at 1562 cm -1 (asym. C=O) and 1431 cm -1 (sym. C=O) for 6d and at 1558 cm -1 (asym. C=O) and 1435 cm -1 (sym. C=O) for 7d. These anilides were prepared as sodium salts in order to keep the amide function stable during the next step which involved the synthesis of the acid chlorides 6e and 7e using thionyl chloride in the presence of 1 equivalent of pyridine (22) , thus avoiding the liberation of HCl. Both 6e and 7e showed a new characteristic FT-IR carbonyl peak (of acid chloride) at 1797 cm -1 with the disappearance of all of the carboxylate peaks.

Scheme 1. Synthesis of the target compounds (6 and 7)
The final step of synthesis involved primary amide formation (6 and 7) by modifying a simple procedure using ice-cooled concentrated aqueous ammonia solution [23] . The FT-IR spectrum showed the disappearance of the 1797 cm -1 peak for both 6 and 7 while the amide carbonyl peaks (1643 cm -1 and 1658 cm -1 , respectively) became broad because of the overlap of the primary and secondary amide carbonyl groups.
The two protons attached to the nitrogen of the primary amide are chemically not equivalent due to hindered amine rotation (24) . This is illustrated by the difference in their chemical shifts in the 1 H-NMR spectrum of 6 and 7 (0.57 ppm and 0.6 ppm respectively).

Cytotoxicity study
Compounds 6 and 7 were tested in vitro for antiproliferative activity against HRT-18 cell line (human colon adenocarcinoma), HC-04 cell line (mouse hepatic carcinoma), and HBL-100 cell line ( epithelial cells obtained from healthy human breast milk) at micromolar concentrations (6.25, 12.5, 25, 50, 100 µM). Both of them showed inhibitory activity against the cancer cell lines higher than against the normal cell line. Figures 3 and 4 show that there is a continuous and parallel increase in the inhibition of growth of both compounds against both colon adenocarcinoma and hepatic carcinoma with increasing the concentration. On the other hand, they show very low cytotoxicity against normal cells.  Figure 6 (captured at the IC50) shows the difference in the magnitude of cytotoxicity between the two compounds.
. The low cytotoxicity of both compounds against normal cells gives hope of being able to target cancer cells to a higher degree than normal cells. No more than 12 % of inhibition of growth was observed at the highest concentration used for both compounds (Figure 7). This might possibly be considered, in agreement with previous reports, that the synthesized compounds might exhibit relative specificity in inhibiting HDACs that are overexpressed in cancer cells.

Conclusion
Synthesis of the target primary amides (6 and 7) was achieved starting from adipic acid (6a) and pimelic acid (7a) respectively . They were fully characterized successfully. Both of them showed good cytotoxicity against cancer cell lines (HC-04 and HRT-18) and lower cytotoxicity against normal cell line (HBL-100).