Effect of 3-subsitution of quinolinehydroxamic acids on selectivity of histone deacetylase isoforms

Abstract A series of 3-subsituted quinolinehydroxamic acids has been synthesised and evaluated for their effect on human lung cancer cell line (A549), human colorectal cancer cell line (HCT116) and HDAC isoforms 1, 2, 6, and 8. The results indicated that substitution at C3 of quinoline is favoured for HDAC6 selectivity. Two compounds (25 and 26) were also found to be potent anti-proliferative compounds with IC50 values ranging from 1.29 to 2.13 µM against A549 and HCT116 cells. These compounds displayed remarkable selectivity for HDAC6 over other HDAC isoforms with nanomolar IC50 values. Western blot analysis revealed that compounds of this series activate apoptotic caspase pathway as indicated by cleavage of caspase 3, 8, and 9 and also increase phosphorylated H2AX thus inducing DNA double strand fragmentation in a concentration dependent manner. Flow cytometric analysis also displayed a dose dependent increase of cell population in sub G1 phase.


Introduction
Over last two decades Epigenetics have emerged as a potential target for the treatment of cancer and various other physiological disorders. Gene expression regulation by epigenetic modifications play pivotal role and thus have drawn a lot interest for the development of therapeutics capable of altering gene expressions by modulating post-translational changes in histone e.g. acetylation, methylation etc. Histone post-translational modifications act as regulatory marks which are important for the control of transcription and chromatin architecture. Chromatin is a compact structure of nucleosomes and is formed from DNA-wrapped histone proteins. Two enzymes, histone acetylase (HAT) and histone deacetylase (HDAC) together control the acetylation level of lysine residues in the chromatin N-terminal region. The acetylation level is correlated with the structure of chromatin. HAT acetylates lysine residues of histone and the resultant neutralised histones loose chromatin, which subsequently results in activation of gene expression. In contrast, HDAC removes acetyl groups and the resulting positively ionised proteins lead to the condensation of chromatin, which represses gene expression. Therefore it can be stated that histone acetylation is carried away by histone transferases (HATs) and histone deacetylases (HDACs) reverse the action of HATs. Epigenetic modification regulates the genetic expression of chromatin without changing its DNA sequence, and the aberrance of this process is highly correlated with the occurrence of disease 1-4,8a . Consequently, epigenetic regulation has currently become an attractive target for the treatment of a variety of diseases such as cancer 5-8b , inflammation, [9][10][11][12][13][14][15][16][17] neurological disorders 18 and HIV [19][20] . Many HDACs have been reported to be overexpressed in various malignancies and therefore various HDAC inhibitors (HDIs) have been developed to treat these diseases. Since 2006, four HDAC inhibitors have been approved by FDA viz. Vorinostat (SAHA), Romidepsin (FK228), Belinostat (PXD101), and Panobinostat (LBH589). SAHA is the first HDI that received FDA approval in 2006 for the treatment of Cutaneous T cell Lymphoma (CTCL). Many new HDAC inhibitors, including dual/multitarget inhibitors 8b , have been developed and many are now in clinical trials ( Figure 1).
Recent reports brought our attention to the development of selective inhibitors and on further modifying the lead molecule (6), we reported the design and synthesis of N-hydroxy-4-((quinolin-8-ylamino)methyl)benzamide (13), a selective HDAC6 inhibitor which exhibits potent activity against multiple myeloma ( Figure 2) 28,29 . Continuing our efforts on investigation of quinoline-containing molecules, the current study focuses on the modification of compound 13, and evaluates the effect of various substituents at C3 of quinoline to generate a series of 3-substituted quinolinehydroxamic acids with selective inhibitory and anti-proliferative activities against HDAC6 (Figure 3).

Western blot analysis
Western blot analysis indicated that compounds of this series induce apoptosis by activation of caspase and PARP pathways.  followed by 26 which was further followed by 17. The order of their efficacy in cleaving caspase isoforms also follows the same order as their in vitro cell growth inhibitory activity with 17 being the least potent amongst these three compounds. These molecules also increase dose dependent expression of gamma H2AX indicating increased DNA double strand fragmentation ( Figure 4).

Flow cytometry analysis
The effects of compound 17, 25, and 26 on cell cycle progression on A549 cells were examined by flow cytometry. Treatment of A549 cells with 25 and 26 resulted in concentration dependent accumulation of A549 cells in sub G1 phase with concomitant losses from the G0/G1 phase. Compounds followed the same trend in their activity as that of western blot analysis owing to same reason. Furthermore, a characteristic hypo diploid sub G0/1-peak was observed as a consequence of increased apoptosis and partial DNA loss in A549 cells treated with 25 or 26 in a dose dependent manner ( Figure 5).

Docking study of compound 17
Compound 17 possesses the most HDAC6 selectivity over other isoforms; therefore, we docked 17 into the published crystal structure of HDAC6 (PDBID: 6CGP, Figure 6) in an attempt to understand the interaction of compound 17 with HDAC6, which was conducted using Discovery Studio2017R2. The hydroxamic acid of 17 interacts with a zinc ion (gray dotted line) and forms a hydrogen bond with His573 (red dotted line) in the bottom of the pocket, which is the typical interaction of hydroxamic acid-containing histone deacetylase inhibitors with HDACs. The central phenyl ring has p-p interactions with Phe583 and Phe643 (violet dotted lines) and the quinoline moiety is able to interact with the surrounding amino acids (His463, Pro464, and Phe583), including p-p (violet dotted lines), p-alkyl (green dotted line), and p-sigma interactions (yellow dotted line). Notably, the C3-methoxy group has an additional carbon hydrogen bond interaction with Asp460 (blue dotted line) in the open area, which probably contributes the higher HDAC6 selectivity.

Conclusion
This study investigated the effect of C3 substitution of quinolinehydroxamic acids on related biological activities, such as antiproliferative and HDAC inhibitory activity. Synthesised compounds showed potent antitumor activities in A549 cells and HCT116 cells with single digit micromolar activities. Pyridine substitution at C3 of quinoline was found to be optimum for the activity of these compounds. The 3-pyridyl compound (25) is more potent than the 4-pyridyl isomer (26) and with IC 50 ¼ 4.75 nM, displayed better HDAC6 selectivity. These compounds exhibit their anticancer effects by induction of apoptosis and by fragmenting DNA double strands as revealed by western blot analysis. The increased 6.34 Â 10 À7 (5) (9) (286) a Dashed line indicates no inhibition or compound activity that could not be fitted to an IC 50 curve. IC 50 value higher than 10 lM is estimated based on the best curve fitting available. b Selectivity ratio: selectivity ratio of HDAC subtypes over HDAC6.  3.84 ± 0.40 3.59 ± 0.51 SAHA [27] 1.02 ± 0.15 0.15 ± 0.03 PXD101 [27] 0.78 ± 0.07 0.13 ± 0.01 Ã IC 50 values higher than 10 lM are estimated based on the best curve fitting available.
population of treated A549 cells in sub G1 phase reveals cell cycle arrest in the sub G1 phase indicating DNA loss and apoptosis. These compounds (17,25,26) are potent and selective HDAC6 inhibitors and are crucial to development of the SAR of quinolinehydroxamic acids and to explore the possibilities of structural modifications to yield compounds with selectivity towards HDAC inhibition.

Chemistry
Nuclear magnetic resonance spectra were obtained with Bruker DRX-300 spectrometer operating at 300 MHz, with chemical shifts reported in parts per million (ppm, d) downfield from TMS, an internal standard. High-resolution mass spectra (HRMS) were measured with a JEOL (JMS-700) electron impact (EI) mass spectrometer. Purity of the final compounds was determined using a Hitachi 2000 series HPLC system using C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 mm. 4.6 mm Â 150 mm) and was found to be ! 95% in all cases. Flash column chromatography was carried out using silica gel (Merck Kieselgel 60, No. 9385, 230-400 mesh ASTM). All reactions were done under an atmosphere of dry nitrogen.
General procedure of halogenation of 8-nitroquinoline (31a-31c): A solution of 8-nitroquinoline (1 eq) in AcOH (10 ml) was stirred at 110 C and N-halosuccinimide (1.1-1.4 eq) was added in portions to the solution. The mixture was stirred at 110 C for 1 h, and then cooled to room temperature (RT). The reaction mixture was poured into H 2 O (50 ml), and the resulting precipitate was collected by filtration and washed with H 2 O. The crude product was purified by flash column chromatography on silica gel eluting with EtOAc/n-Hexane to afford desired compound.
General Procedure of reductive amination (32-36): A mixture of nitro compound (1 eq), iron powder (4 eq), NH 4 Cl (5 eq), and 75% MeOH (10 ml) was heated to reflux. The reaction mixture was filtered and the filtrate was concentrated in vacuo, and then extracted with DCM and H 2 O. The combined organic layer was dried over anhydrous MgSO 4 , and concentrated in vacuo to afford a crude residue. A mixture of the residue (1 eq), methyl 4-formylbenzoate (1.2 eq), sodium triacetoxyborohydride (1.5 eq), AcOH (2-4 drops), and anhydrous DCM (10 ml) was stirred at RT. The mixture was poured into H 2 O, and then extracted with DCM. The combined organic layer was purified by flash column chromatography on silica gel with EtOAc/n-Hexane to afford corresponding ester compound 32-36.
General procedure for Suzuki arylation (37-47): A mixture of 33 (1 eq), substituted phenylboronic acid (1.2 eq), tetrakis(triphenylphosphine)palladium(0) (0.1 eq), TBAB (0.4 eq), 2 M K 2 CO 3 (1 ml) and dioxane (10 ml) was stirred and refluxed overnight. Dioxane was removed from the reaction mixture in vacuo, and the mixture was extracted with H 2 O and EtOAc. The organic layer was collected and concentrated into an oily residue which was purified via column chromatography on silica gel with EtOAc/n-hexane to afford corresponding esters 37-47. General procedure for hydroxamic acid synthesis. A mixture of 1 N LiOH (3 ml) and ester (1 mmol) was stirred at 40 C for 2 h. The reaction was concentrated under reduced pressure and then H 2 O was added. The mixture was acidified with 3 N HCl to give an offwhite precipitate. This off-white solid (1 mmol) was dissolved in DMF (10 ml) and EDCÁHCl (1.5 mmol) was added, followed by HOBt hydrate (1.5 mmol) and TEA (3 mmol). After being stirred at RT for 30 min, NH 2 OTHP (1.2 mmol) was added and allowed to stir for an additional 5 h. The reaction mixture was quenched with H 2 O and was extracted with EtOAc (25 ml Â 3). The combined organic layer was collected, dried over anhydrous MgSO 4 and concentrated under reduced pressure to give a light yellow residue, which was purified by silica gel chromatography (EtOAc: n-hexane ¼ 1:1) to give a colourless liquid. 10% TFA (5 ml) was added to the resulting product dissolved in MeOH (5 ml) and the mixture was stirred at RT for 5 h. The reaction mixture was concentrated under reduced pressure to give a white residue, which was recrystallized from MeOH to afford the desired compound.

3-Methoxy-8-nitro-quinoline (31d)
A mixture of 31c (0.10 g, 0.33 mmol), CuI (0.006 g, 0.03 mmol), 1,10-phenanthroline (0.011 g, 0.06 mmol), Cs 2 CO 3 (0.16 g, 0.50 mmol), MeOH (1 ml), and toluene (0.5 ml) was placed in a 10 ml reaction vessel with a magnetic stirring bar. The vessel was sealed and placed in the microwave cavity. The reaction condition was held at 70 C for 30 min. After it was cooled to RT, the reaction mixture was filtered and the filtrate was concentrated in vacuo, and then extracted with DCM and H 2 O. The combined organic layer was purified by flash column chromatography on silica gel with EtOAc/n-hexane to afford compound 31d (0.04 g, 57%): 1

The sulforhodamine B assays
Cells were seeded at the density of 5000 cells/well into 96-plate overnight. Basal cells were fixed with 10% trichloroacetic acid (TCA) to represent the cell population at the time of compound addition (T 0 ). After additional incubation of DMSO (C) or different doses of test compounds (Tx) for 48 h, cells were fixed with 10% TCA and stained with SRB at 0.4% (w/v) in 1% AcOH. Unbound SRB was washed out using 1% AcOH and SRB bound cells were solubilised with 10 mM Trizma base. The absorbance was read at a wavelength of 515 nm. The 50% growth inhibition (GI 50 ) was calculated by 100 À [(T x -T 0 )/(C -T 0 )] Â 100.

HDAC biochemical assays
The HDACs in vitro activities of human recombinant HDAC 1, 2, 4, 6, and 8 were conducted by EurofinPanlabs (Taipei, Taiwan). In brief, indicated compounds were incubated with specific HDAC enzyme and Fluor-de-Lys deacetylase substrate. Fluor-de-Lys deacetyl substrates were spectrofluorimetrically quantitated compared to control.

Western blot analysis
Cells were incubated with indicated compounds for 24 h and lysed with ice-cold lysis buffer (20 mMTris-HCl pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 2.5 mM b-glycerylphosphate, 1 mM Na 4 P 2 O 7 , 5 mM NaF, 1 mM Na 3 VO 4 and protease inhibitor cocktail from Millipore) on ice for 30 min following by centrifugation at 13000 rpm for 30 min. Protein concentrations were determined and equal amounts of protein were separated by 8-15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly(vinylidene difluoride) (PVDF) membranes. Membranes were immunoblotted with specific antibodies overnight at 4 C and then applied to appropriate horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies for 1 h at RT. Signals were detected using an enhanced chemiluminescence (Amersham, Buckinghamshire, UK).

Flow cytometry
Briefly, A549 cells were starved with EBM-2 medium overnight and were subsequently replenished with EGM-2 medium with or without 17, 25, and 26 (0.25, 0.5, 10 mM) for 48 h. After being trypsinized and fixed in ice-cold 75% MeOH for 1 h at À20 C, A549 cells were washed with PBS and resuspended in 0.2 ml DNA extraction buffer (0.2 M Na 2 HPO 4 , 0.1 M citric acid; pH 7.8) for 30 min. Then the cells were stained with propidium iodide solution (PI; 100 mg/ml RNase, 80 mg/ml propidium iodide, 0.1% Triton X-100) in PBS. FACScan flow cytometry was utilised to determine cell cycle distribution, and data analysis was performed with CellQuest software (BD Biosciences).

Statistical and graphical analyses
Each experiment was performed independently at least three times and the data are presented as mean ± SEM for the indicated number of separate experiments. Student's t-test was used to compare the mean of each group with that of the control group in experiments and one-way ANOVA was used in animal study. p Values <0.05 were considered significant ( Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001).

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This research was supported by the Ministry of Science and Technology, Taiwan [grant no. MOST108-2320-B-038-042-MY3].