Discovery of potent histone deacetylase inhibitors with modified phenanthridine caps

Abstract In discovery of novel HDAC inhibitory with anticancer potency, pharmacophores of phenanthridine were introduced to the structure of HDAC inhibitors. Fatty and aromatic linkers were evaluated for their solubility and activity. Both enzyme inhibitory and in vitro antiproliferative (against U937 cells) screening results revealed better activities of compounds with aromatic linker than molecules with fatty linker. Compared with SAHA (IC50 values of 1.34, 0.14, 2.58, 0.67 and 18.17 µM), molecule Fb-4 exhibited 0.87, 0.09, 0.32, 0.34 and 17.37 µM of IC50 values against K562, U266, MCF-7, U937 and HEPG2 cells, respectively. As revealed by cell cycle and apoptotic analysis, induction of G2/M phase arrest and apoptosis plays an important role in the inhibition of MCF-7 cells by Fb-4. Generally, a potent HDAC inhibitor was developed in the present study which could be utilised as a lead compound for further anticancer drug design.


Introduction
Histone deacetylases (HDACs) are a group of enzymes that are responsible for the deacetylation E-N-acetyl-lysine amino groups on histones and various non-histone proteins 1,2 . Until now, 18 isoforms of HDACs which are divided into 4 classes have been identified in human 3,4 . Class I HDACs (HDAC1, 2, 3 and 8) have been extensively studied in the field of anticancer drug development. Class II HDACs are subdivided into IIa (HDAC4, 5, 7 and 9) an IIb (HDAC6 and 10). Class III HDACs are a group of NAD þ dependent enzymes which are also termed sirtuins (Sirt1-7). There is only one member in class IV, HDAC11, which is the most recently identified and smallest known HDAC enzyme.
The modification of histone and non-histone proteins by HDACs and histone acetyl transferases (HATs) plays an important role in the regulation of cellular functions 5 . Overexpression and aberrant recruitment of HDACs (especially class I and II HDACs) are closely related to tumorigenesis and tumour development 6,7 . Inhibition of HDACs has been extensively investigated for the treatment of cancer. Various types of HDAC inhibitors have been designed and synthesised for the discovery of anticancer drugs 8 . The first US FDA approved HDAC inhibitor, Vorinostat (SAHA) 9 , has been utilised for the treatment of cutaneous T-cell lymphoma (CTCL). Following the success of SAHA, romidepsin (FK-228), belinostat (PXD101), and panobinostat (LBH589) were approved for the treatment of CTCL, peripheral T-cell lymphoma (PTCL), and multiple myeloma [10][11][12] , respectively.
There is a narrow tunnel in the active site of HDACs, and a zinc ion locates at the end of the tunnel. At the opening of the active site, according to different isoforms of HDACs, there are binding pockets with various shape and size. Generally, pharmacophores of HDAC inhibitors are consist of a zinc binding group (ZBG), a linker and a cap 8 . Class I, II and IV HDACs are zinc dependent enzymes, and there is a zinc ion in the active site of these HDACs. Therefore, ZBGs are needed in the structure of HDAC inhibitors for the binding of inhibitors to the active site. Cap region binds to the opening of the active site, aromatic groups in this region could form strong hydrophobic interactions with surrounding residues. Linker which is used to connect cap and ZBG can also interact with residues in the active site of HDACs.
In our previous studies, a series of phenanthridine derivatives were developed with high anticancer activities by targeting DNA topoisomerase 13 . However, the synthesised molecules are extremely low at both aqueous solubility and liposolubility. Moreover, in the in vivo studies using xenograft nude mice model, molecule 8a exhibited obvious hepatotoxicity. Considering the aromatic properties of phenanthridine structure, strong hydrophobic interactions could be formed between phenanthridine fragment and residues in the opening of HDAC active site. Therefore, in discovery of anticancer agents with improved solubility, activity and safety profiles, pharmacophores of phenanthridine was introduced to the cap region in the structure of HDACIs ( Figure 1). By targeting HDACs, the toxicity of the designed molecules was considered to be reduced. To decrease the aromaticity of the designed compounds, the B ring in the phenanthridine structure was opened for the introduction of substituents. Hydroxamic acid group was utilised as zinc binding group, aromatic and fatty linkers were introduced, respectively. The synthesised target compounds were investigated in the enzyme inhibitory assay, in vitro antiproliferative screening, cell cycle and apoptosis test.

Chemistry
The target molecules were synthesised as illustrated in scheme 1. At first, the amino group of the starting material benzo[d] [1,3]dioxol-5-amine was protected by Boc group. The following bromine substitution was carried out to generate intermediate 3.

Enzymatic inhibition assay
The HDAC enzyme inhibitory activities of synthesised compounds were investigated by utilising Hela nucleus extract containing a mixture of HDAC isoforms. Percentage inhibitory rate was used to determine the activity of tested compounds (Tables 1 and 2). The Fa series compounds were firstly synthesised for the activity screening. The results revealed that all the compounds with fatty linker are less active compared with the positive control SAHA at concentration of 1 mM. To investigate whether aromatic linker could improve the inhibitory activities, structural modifications were performed on current molecules. The derived Fb series compounds with aromatic linker showed improved HDAC inhibitory activities compared with the Fa series molecules. Molecule Fb-2, Fb-3 and Fb-4 exhibited inhibitory rate of 52.30, 53.90 and 66.54, respectively, compared with SAHA (inhibitory rate of 56.23).
Different subtypes of HDACs may play different biological effects in cells. Therefore, it is important to determine the inhibitory selectivity of active HDAC inhibitors. In the present study, the selectivity of representative compound Fb-4 was tested against HDAC1, 2, 3, 6 and 8 ( Table 3). The results revealed that compound Fb-4 has no obvious HDAC isoform selectivity in the inhibition of HDAC1 (IC 50 value of 12.1 nM), HDAC2 (IC 50 value of 21.5 nM), HDAC3 (IC 50 value of 11.0 nM) and HDAC6 (IC 50 value of 8.6 nM). Whereas, in the inhibition of HDAC8, compound Fb-4 exhibited reduced potency with IC 50 value of 304 nM. It is indicated that molecule Fb-4 is a pan HDAC inhibitor.

Antiproliferative activity
All the synthesised molecules were screened against U937 cells (Tables 1 and 2), and only active molecules which exhibited good performances in both enzymatic and antiproliferative test were selected for the cancer cell growth inhibitory assay against various cell lines. In the U937 cell based screening, The Fb series molecules with mean inhibitory rate of 77.32 also showed significantly improved activities compared with the Fa series molecules (with mean inhibitory rate of 20.82). Obviously, compound Fb-2, Fb-3       molecule Fb-4 could inhibit cell proliferation of MCF-7 cells by inhibiting protein synthesis and rapid cell growth as a result of G2/M phase arrest.

Apoptotic analysis
Apoptosis plays an important role in the treatment of cancer. Moreover, efforts to increase G2/M arrest have also been associated with enhanced apoptosis. Therefore, to determine whether the cell cycle arrest caused by molecule Fb-4 can ultimately induce apoptosis, MCF-7 cells were treated with different concentrations of molecule Fb-4 (1, 3 and 9 mM). As shown in Figure 3, increased number of apoptotic cells was detected in MCF-7 cells treated with various doses of Fb-4 (3 A) and SAHA (3B). It is significant that both Fb-4 and SAHA induced MCF-7 cell apoptosis in a dose dependent manner. After treatment with difference concentrations of Fb-4 (1, 3 and 9 mM), the percentage of apoptotic cells were significantly increased from 9.16% of the control to 16.99%, 20.07% and 21.19%, respectively, compared with SAHA (11.38%, 12.30% and 19.51% at the concentration of 1, 3 and 9 mM, respectively). It is indicated that induction of cell apoptosis makes contributions to the anti-proliferative effect of compound Fb-4.

Conclusion
In discovery of lead compound for the anticancer drug development, pharmacophores of phenanthridine structure were introduced to the cap region of HDAC inhibitors. Two series of novel HDAC inhibitors were synthesised with different linkers. In the enzyme inhibitory and in vitro antiproliferative screening, target compounds with aromatic linker exhibited improved activities compared with molecules with fatty linker. In the in vitro cancer cell based test, the selected compounds showed potency in the inhibition of both solid (MCF-7 and HEPG2 cells) and haematologic (K562, U266 and U937 cells) tumour cell lines compared with SAHA. Significantly, compared with SAHA, molecule Fb-4 displayed 0.87, 0.09, 0.32, 0.34 and 17.37 mM of IC 50 values against K562, U266, MCF-7, U937 and HEPG2 cells, respectively. Cell cycle and apoptotic analysis revealed that induction of G2/M phase arrest and apoptosis relate to the antiproliferative potency of Fb-4. Collectively, a potent lead compound (Fb-4) was discovered for the treatment of cancer by inhibition of HDACs. It must be pointed out that molecules with aromatic linker have poor solubility in both aqueous and lipid solutions. Therefore, structural modification of compound Fb-4 will be performed by improving the pharmacokinetic profiles and anticancer potency in our further research.

Materials and methods
All chemicals were obtained from commercial suppliers and can be used without further refinement. All reactions were detected by TLC using 0.25 mm silica gel plate (60GF-254). UV light and ferric chloride were used to show TLC spots. Due to the poor solubility of the target compounds, only the 1 H NMR spectra were derived for the structural identification. 1 H NMR spectra were recorded on a Bruker DRX spectrometer at 500 MHz, using TMS as an internal standard.  Compound 1-3 were synthesised as described in our previous work 13 .

Synthesis of compound 6a
Compound 6a (0.4 g, 0.88 mmol) was dissolved in dry DCM. After addition of TFA, the solvent was stirred at room temperature, monitored by TLC. Until the raw materials were completely consumed, the reagents were evaporated under vacuum and dissolved with a mixed solution of tetrahydrofuran and water (50:1) at 0 C with addition of sodium bicarbonate. After stirred for 10 min, 3-Bromobenzoyl chloride (0.23 g, 1.03 mmol) was added, and the misture was stirred at room temperature for 4 h. After that, the reagents were evaporated under vacuum and dissolved in EtOAc. The solvent was washed with saturated NaHCO 3 (3 Â 20 ml) and brine (1 Â 20 ml), dried over MgSO 4 , and evaporated under vacuo. The desired compound fa-1 (0.43 g, 91% yield) was derived by crystallisation in EtOAc as white powder.

In vitro HDACs inhibitory assay
All HDAC enzymes were purchased from BPS Bioscience. In short, 60 lL of recombinant HDAC enzyme solution was mixed with various concentrations of test compound (40 lL), and then incubated at 37 C for 30 min. The reaction was terminated by adding 100 lL of imaging agent containing trypsin and trichostatin A (TSA). After standing for 20 min, the fluorescence intensity was measured at the excitation and emission wavelengths of 360 and 460 nm with a microplate reader. The inhibition rate was calculated from the fluorescence intensity readings of the test wells relative to the control wells, and the IC 50 curve and value were determined by GraphPad Prism 6.0 software.

In vitro antiproliferative activity
SAHA was used as a control and the MTT assay was used to determine tumour cell suppression. K562, U266, MCF-7, U937 and HEPG2 cells were cultured in corresponding medium supplemented with 10% FBS. Dilute the stock solution of the test compound with the culture medium. In short, cells were seeded into each well of a 96-well plate and incubated at 37 C under 5% CO 2 . The cells were then treated with various concentrations of compound samples for 48 h. After that, add 10 ll MTT working solution to each well and incubate for another 4 h. After incubation, the medium formed by MTT was extracted by adding DMSO (100 mL). Measure the absorbance (OD) at 570 nm and 630 nm with a microplate reader. The cell growth inhibition rate was calculated according to the following formula:% inhibition ¼ [1-(Sample group OD 570 -Sample group OD 630 )/(Control group OD 570 -Control group OD 630 )] Â 100%. Use Origin 7.5 software to calculate IC50 value.
Cell cycle assay MCF-7 cells in the logarithmic growth phase were seeded in a 6well plate and incubated with different doses of Fb-4 and SAHA (1, 3, and 9 lM) for 24 h. The cells were then washed twice with cold PBS and fixed in 70% pre-chilled ethanol at 4 C for 6 h. After fixation, the cells were washed again with PBS and stained with PI/RNase A at 37 C for 30 min, and then stored at 4 C in the dark. After staining, the cell cycle distribution was determined by flow cytometry within 24 h.
Cell apoptosis assay MCF-7 cells in the logarithmic growth phase were seeded in 6well plates and incubated with different doses of Fb-4 and SAHA (1, 3 and 9 mM) for 24 h. After that, wash the cells with PBS and collect the cells, resuspend them in the binding buffer of the Annexin V-FITC kit, then add 5 lL of Annexin V-FITC and mix gently, and then place it at 2-8 C in the dark Incubate. After incubating for 15 min, add 10 mL of PI to each sample and mix gently, and incubate for 5 min at 2-8 C under dark conditions, and detect with a flow cytometer.

Author contributions
Lei Zhang designed the project. Wenli Fan and Haiyong Jia synthesised the molecules; Lin Zhang performed the enzymatic screening; Xuejian Wang performed the in vitro antitumor experiments. Lei Zhang and Wenli Fan analysed the date and wrote the manuscript.

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