Design, synthesis, and preliminary bioactivity evaluation of N1-hydroxyterephthalamide derivatives with indole cap as novel histone deacetylase inhibitors

Article abstract of DOI:10.1111/cbdd.12819

Histone deacetylases inhibitors (HDACIs) have been widely recognized as significant therapeutic approach to cancers. In our efforts to develop novel histone deacetylases inhibitors (HDACIs) as potential anticancer agents, a series of N¹-hydroxyterephthalamide derivatives with an indole cap group were designed and synthesized. Compound 12m was identified to be the most potent one (IC₅₀?=?0.074?μm against HeLa nuclear extract) and showed higher inhibitory activity than the positive control SAHA (IC₅₀?=?0.131?μm), which was also verified by further molecular docking studies into active site of HDAC2. The results of selectivity on the inhibition of HDACs exhibited 12m being with similar isoform selective profile with PXD101. In addition, the representative compounds (8d, 12d, 12j, 12m) based on the outcomes of preliminary tumor cell screening demonstrated more potent or comparable to SAHA in the next antiproliferative activity assays. Collectively, the results encouraged further development of this chemical template to provide more potent analogs as HDACIs.

Full text of DOI:10.1111/cbdd.12819

Received Date : 30-Apr-2016
Revised Date : 28-Jun-2016
Accepted Date : 02-Jul-2016
Article type
: Research Article
Design, synthesis and preliminary bioactivity evaluations of
N1-hydroxyterephthalamide derivatives with indole cap as novel histone
deacetylase inhibitors
Xue Wang a, Xiaoyang Li a, Jingyao Li a, Jinning Hou a, Ying Qu a, Chenggong Yu a, Feng He
a, Wenfang Xu a, Jingde Wu a*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44
West Culture Road, 250012 Ji’nan, Shandong, PR China
Corresponding author, Tel: +86-531-88382006; Fax:+86-531-88382006, E-mail:
wujingde70@sdu.edu.cn, jingdewu70@gmail.com.
Abstract
Histone deacetylases inhibitors (HDACIs) have been widely recognized as significant
therapeutic approach to cancers. In our efforts to develop novel histone deacetylases
inhibitors (HDACIs) as potential anticancer agents, a series of N1-hydroxyterephthalamide
derivatives with an indole cap group were designed and synthesized. Compound 12m was
identified to be the most potent one (IC50=0.074μM against Hela nuclear extracts) and
showed higher inhibitory activity than the positive control SAHA (IC50=0.131μM), which
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.12819
This article is protected by copyright. All rights reserved.

was also verified by further molecular docking studies into active site of HDAC2. The results
of selectivity on the inhibition of HDACs exhibited 12m being with comparable similar
overall isoform selective profile with PXD101. In addtion, the representative compounds
based on the outcomes of preliminary tumor cell screening demonstrated more potent or
comparable to SAHA in the next antiproliferative activity assaies. Collectively, the results
encouraged further development of this chemical template to provide more potent analogs as
HDACIs.
Keywords: HDACIs, molecular docking, antiproliferative activity, selectivity
1. Introduction
The epigenetic abnormality is determined by chromatin structure including DNA
methylation, histone variants and modifications, nucleosome remodeling as well as small
non-coding regulatory RNAs, and viewed as another pathway to initiation and progression of
cancer based on genetic mutations1. Histone deacetylases (HDACs), exert silencing effects on
cell-cycle regulatory genes associated with cell proliferation, differentiation and diminishing
apoptosis which play a crucial role in the transition of normal cells into malignant cells2.
Thus far, 18 HDACs have been identified in humans and categorized into four classes due to
their sequence homology to yeast orthologs3. Class I, including HDACs 1, 2, 3 and 8, shares
homology with Rpd3. Class II can be divided into two subclasses – IIa (HDACs 4, 7 and 9)
and IIb (HDACs 6 and 10), which holds homology to HDAC1. Class IV, covers the sole
member of HDAC11 and possesses homology with both class I and class II. The 11 family
members referred to as classical HDACs, are metal-dependent enzymes inhibited by Zn2+ ion
chelating compounds with differ in the catalytic mechanisms of class III HDACs as these
enzymes with homology to the Sir2 (Silent information regulator 2) family requiring NAD+
as an essential cofactor during catalysis4. Although the biological functions of HDAC
isoforms have not been fully understood yet, HDACs expression in specific tissues as well as
This article is protected by copyright. All rights reserved.

organs, and their different levels in various types of cancers both contribute to indicate the
progress of diseases and develop agents with more precise therapeutic effects5.
Up to date, four HDACIs, vorinostat (SAHA)6, romidepsin (FK228) 7, belinostat
(PXD101)8,9 and panobinostat (LBH589)10 have been approved by the US FDA. SAHA and
FK228 are approved for the clinical therapy of cutaneous T-cell lymphoma (CTCL), PXD101
is for the treatment of peripheral T-cell lymphoma (PTCL) and LBH589 is applied for
combination therapy with bortezomib and dexamethasone for the treatment of recurrent
multiple myeloma11. Additionly, over 20 HDACIs were in diverse clinical trials during the
past 10 years. In general, numerous HDACIs share a common pharmacophore model which
consists of three portions: zinc ion binding group (ZBG) that chelates zinc ion of HDACs
active site; hydrophobic opposite capping group that enhances the affinity with residues of
external HDACs and a linker, connecting ZBG and cap group (Fig. 1) 12.
Olson and co-workers reported that compound 2b13 exhibited potent HDACs inhibitory
activity and excellent selectivity for HDAC2 (IC50=0.61 μM) and HDAC6 (IC50=0.004 μM),
thus suggesting that the N1-hydroxyterephthalamide could be an ideal moiety in the design of
effective HDACIs. Indole structure appears frequently in molecule drug design14, 15 and has
been successfully employed in design of potent HDACIs such as the FDA-approved
panobinostat and Quisinostat that is under clinical trial (JNJ-26481585)16,17. Our laboratory is
also interested in exploring the indole-containing cap group in designing novel HDACIs. For
example, compound 14a18 exhibited potent in vitro and in vivo anti-cancer activities. In
continuation of our efforts to explore this structural feature and design of new HDACIs,
herein, we reported the design, synthesis and preliminary bioactivity evaluation of
N1-hydroxyterephthalamide derivatives with indole cap as HDACIs (Fig. 2). Preliminary
structure-activity relationship (SAR) studies were also discussed.
This article is protected by copyright. All rights reserved.

2. Experimental section
2.1 Chemistry
All commercially available starting materials, reagents and solvents were used without
further purification unless otherwise stated. All reactions were monitored by TLC using 0.25
mm silica gelplates (60GF-254). UV light and ferric chloride were used to visualize the spots.
1H NMR spectra were obtained on a Bruker DRX spectrometer at 400 MHz or 300 MHz with
TMS as an internal standard, δ in parts per million and J in hertz. High-resolution mass
spectrometry was performed by Shandong Analysis and Test Center in Ji’nan, China.
ESI-MS spectra were recorded on an API 4000spectrometer. Silica gel was used for column
chromatography purification. Flashchromatography was accomplished using the automated
Combi Flash Rf system from Teledyne ISCO and was done using silicagel of 200-300 mesh.
Melting points were determined on an electro thermal melting point apparatus.
2.1.1 General Procedure for the Preparation of 2
(Tert-Butoxycarbonyl)-L-tryptophan (2)
L-Tryptophan (10.2 g, 50 mmol) was dissolved in 1 M NaOH (100 mL) aqueous solution,
then solution of Boc2O (13.08 g, 60 mmol) in THF was added dropwise. After the reaction
finished, THF was evaporated. The residual aqueous solution was acidified with 1 N HCl
until pH 3-4 and then extracted with EtOAc (3×100 mL). The organic layer was washed with
brine (2×50 mL) and dried over Na2SO4 overnight, then evaporated under vacuum to
obtained white solid 2, which was directly used in the next step without further purification.
This article is protected by copyright. All rights reserved.

2.1.2 General Procedure for the Preparation of 3
(S)-benzyl 2-((tert-butoxycarbonyl)amino)-3-(1H-indol-3-yl)propanoate (3)
To a solution of 2 (9 g, 30 mmol) in anhydrous CH2Cl2 (150 ml), was added HOBT (4.8 g, 36
mmol) and EDCI (6.9 g, 36 mmol) at 0ºC, followed by Et3N (3.6 g, 36 mmol). After 30 min,
phenylmethanol (3.9 g, 36 mmol) was added 30 min later. After 9 h, the solution of CH2Cl2
was washed with 1 M HCl (2×40 mL), saturated Na2CO3 (2×40 mL) and brine (2×40 mL),
dried over MgSO4 overnight, and the solvent was evaporated under vacuum to afford white
solid 3, which was directly used in the next step without further purification.
2.1.3 General Procedure for the Preparation of 4
(S)-Benzyl 2-amino-3-(1H-indol-3-yl) propanoatehydrochloride (4)
Compound 3 (9.9 g, 25 mmol) was dissolved in a solution of EtOAc (100 mL) saturated by
dry HCl gas. The solution was stirred at room temperature overnight. The filtered precipitate
was washed by diethyl ether to give the white solid powder 4, which was directly used in the
next step without further purification.
2.1.4 General Procedure for the Preparation of 5
(S)-Methyl-4-((1-(benzyloxy)-3-(1H-indol-3-yl)-1-oxopropan-2-yl) carbamoyl) ben-zoate (
5)
To a solution of 4-(methoxycarbonyl)benzoic acid (3.6 g, 20 mmol) in anhydrous CH2Cl2,
was added 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU,
7.7 g, 24 mmol), followed by Et3N (2.4 g, 24 mmol). After 30 min, compound 4 (6.6 g, 20
mmol) was added followed by Et3N (2.4 g, 24 mmol). 4 h later, the solution of CH2Cl2 was
washed with 1 M HCl (2×40 mL), saturated Na2CO3 (2×40 mL) and brine (2×40 mL), dried
This article is protected by copyright. All rights reserved.

over MgSO4 overnight and evaporated under vacuum. The desired compound 5 was derived
by crystallization in EtOAc as white powder.
2.1.5 General Procedure for the Preparation of 6
(S)-3-(1H-indol-3-yl)-2-(4-(methoxycarbonyl) benzamido) propanoic acid (6)
Compound 5 (6.8 g, 15 mmol) was dissolved in 200 mL MeOH, and then Pd/C of 0.68 g was
added. The solution was stirred at room temperature overnight under the atmosphere of
hydrogen, then was filtered with diatomite, evaporated under vacuum and washed by diethyl
ether to give white solid 6, which was directly used in the next step without further
purification.
2.1.6 General Procedure for the Preparation of 7b-h
(S)-Methyl-4-((3-(1H-indol-3-yl)-1-oxo-1-(propylamino) propan-2-yl) carbamoyl) benzoate
(7b)
To a solution of compound 6 (0.73 g, 2 mmol) in anhydrous CH2Cl2 (20 ml), was added
propan-1-amine (0.12 g, 2 mmol), Benzotriazol-1-yl-oxytripyrrolidino- phosphonium
hexafluorophosphate (PyBop,1.0 g, 2 mmol) at 0ºC, followed by Et3N (0.2 g, 2 mmol). The
solution was stirred at room temperature overnight. The solution was washed with 1 M HCl
(2×10 mL), saturated Na2CO3(2 ×10 mL) and brine (2×10 mL), dried over MgSO4 overnight
and the solvent was evaporated under vacuum. The desired compound 7b was derived by
crystallization in mixture of EtOAc and petroleumether as white solid.
Compounds 7c-h were prepared using the same procedure as described above.
This article is protected by copyright. All rights reserved.

2.1.7 General Procedure for the Preparation of 8a-h and 12a-p
(S)-N1-(3-(1H-indol-3-yl)-1-oxo-1-(propylamino) propan-2-yl)-N4-hydroxyterephthalamide
(8a)
KOH (38.64 g, 690 mmol) and NH2OH HCl (23.35 g, 343 mmol) were dissolved,
respectively, in 70 mL and 120 mL MeOH to get solution A and solution B. Next solution A
was added drop wise to solution B. After filtering the precipitate (KCl), a NH2OK solution
was obtained. Compound 6 (0.38 g, 1 mmol) was dissolved in the NH2OK solution and
stirred overnight. After the reaction was completed, it was evaporated under vacuum. The
residue was acidified with 1 N HCl to a pH 3-4 and then extracted with EtOAc (3×20 mL).
The organic layer was washed with brine
(2×30 mL) and dried over Na2SO4 overnight. The crude material was purified via flash
chromatography to afford the light brown solid 8a.
Compounds 8b-h and 12a-p were prepared using the same procedure as described above.
2.1.8 General Procedure for the Preparation of 9a-h
(S)-tert-butyl (3-(1H-indol-3-yl)-1-oxo-1-(phenylamino)propan-2-yl)carbamate (9a)
Compound 2 (3.0 g, 10 mmol) was dissolved inanhydrous CH2Cl2,
2-(1H-Benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluronium tetrafluoroborate (TBTU, 3.9 g, 12
mmol) was added, followed by Et3N (1.2 g, 12 mmol). After 30 min, aniline (1.1 g, 12 mmol)
was added. 4 h later, the solution of CH2Cl2 was washed with 1 M HCl (2 ×20 mL), saturated
Na2CO3 (2×20 mL) and brine (2×20 mL), dried over MgSO4 overnight, and the solvent was
evaporated under vacuum, affording white solid 9a, which was directly used in the next step
without further purification.
Compounds 9b-h were prepared using the same procedure as described above.
This article is protected by copyright. All rights reserved.

2.1.9 General Procedure for the Preparation of 10a-h
(S)-2-amino-3-(1H-indol-3-yl)-N-phenyl-propionamide hydrochloride (10a)
Compound 9a (3.0 g, 8 mmol) was dissolved in a solution of EtOAc (30 mL) saturated by dry
HCl gas. The solution was stirred at room temperature overnight. The filtered precipitate was
washed by EtOAc to give white solid powder 10a, which was directly used in the next step
without further purification.
Compounds 10b-h were prepared using the same procedure as described above.
2.1.10 General Procedure for the Preparation of 11a-h
(S)-Methyl-4-((3-(1H-indol-3-yl)-1-oxo-1-(phenylamino) propan-2-yl) carbamoyl) benzoat
e (11a)
To a solution of 4-(methoxycarbonyl)benzoic acid (0.9 g, 5 mmol) in anhydrous CH2Cl2, was
added 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1.9
g,6 mmol), followed by Et3N (0.6 g, 6 mmol). After 30 min, compound 10a (1.6 g, 5 mmol)
was added followed by Et3N (0.6 g, 6 mmol). The solution was stirred at room temperature
overnight, then washed with 1 M HCl (2 ×20 mL), saturated Na2CO3 (2×20 mL) and brine
(2×20 mL), dried over MgSO4 overnight, and the solvent was evaporated under vacuum to
afford white solid 11a, which was directly used in the next step without further purification.
Compounds 11b-h were prepared using the same procedure as described above.
2.2 In vitro HDACs inhibition fluorescence assay
In vitro HDACs inhibition assays were conducted as previously described16. In brief, 10
μL of Hela nuclear extracts was mixed with various concentrations of target compounds (50
μL), SAHA, using 100% and none HDACs groups as control group, and the mixture. After
incubation at 37ºC for 10 min, fluorogenic substrate Boc-Lys (acetyl)-AMC (40 μL) was
This article is protected by copyright. All rights reserved.

added and then the mixture was incubated at 37ºC for 30 min. The mixture was stopped by
addition of 100 μL of developer containing trypsin and TSA afterwards. Over the next
incubation at 37ºC for 20 min, fluorescence intensity was measured using a microplate reader
at excitation and emission wavelengths of 390 nm and 460 nm, respectively. The inhibition
ratios were calculated from the fluorescence intensity readings of tested wells relative to
those of control wells, and the IC50 values were calculated using a regression analysis of the
concentration/inhibition data.
2.3 Molecular Docking Studies
Compounds were docked into the active site of HDAC2 (PDB entry: 4LXZ) using Tripos
sybyl x 2.0 Before the docking process, the protein structure was treated by deleting water
molecules, adding hydrogen atoms, fixing atom types, and assigning AMBER7 FF99
charges. A 100-step minimization process was performed to further optimize the protein
structure. The molecular structures were generated with the Sybyl/Sketch module and
optimized using Powell’s method with the Tripos force field with convergence criterion set at
0.05 kcal/(Å mol) and assigned charges with the Gasteiger_Hückel method. Molecular
docking was carried out via the Sybyl/FlexX module. Other docking parameters were kept to
the default values.
2.4 In vitro antiproliferative assay
In vitro antiproliferative assays were determined by the MTT
(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) method as previously
described16. All cell lines were maintained in RPMI1640 medium containing 10% FBS at
37ºC in a 5% CO2 humidified incubator. Cell proliferation assay was determined by the MTT
method. Briefly, cells were passaged the day before dosing into a 96-well cell plate, allowed
to grow for 12 h, and then treated with different concentrations of compound sample for48 h.
A 0.5% MTT solution was added to each well. After incubation for another 4 h, formazan
This article is protected by copyright. All rights reserved.

formed from MTT was extracted by adding 150 μl of DMSO rocking for 15 min. Absorbance
was then determined using an ELISA reader at 570 nm.
3. Results and Discussion
3.1 Chemistry
Given the reports above, we decided to keep N1-hydroxyterephthalamide with indole cap
group as active structural fragments and designed compounds with different substituents,
mainly including aliphatic amine and aromatic amine, to evaluate the impact of various
functional groups on the inhibitory activities on HDACs. The synthesis of target compounds
8a-h was described in Scheme 1. Briefly, the starting compound of L-tryptophan gave 2 by
Boc-protection. Compound 2 was reacted with benzyl alcohol to give the ester intermediate
319. Deprotection followed by the coupling reaction with mono-methyl terephthalate in the
presence of TBTU afforded 5. Hydrogenation in the presence of Pd/C followed by ester
amide exchange reaction produced target compound 8a. On another route, coupling of 6 with
various aliphatic amines followed by ester amide exchange reaction with acid yielded 8b-h in
good yield20. The synthesis of 12a-p followed similar reactions to the synthetic method for
compounds 8a-h.
3.2 HeLa cell extracts inhibition of the target compounds
As shown in the Table 1, within the aliphatic amide analogs, the chain length seemed to
impact the potency dramatically with the shorter chain analog (8b) being more potent.
Compounds 8e-h exhibited comparable potent on the inhibition of HDACs and this might
suggest that steric or hydrophobic ininteractions were preferred at this specific position. The
results of compounds 12b and 12c also supported this notion by showing sub-micromolar
potency. Notably, aromatic amide analogs overall exhibited better inhibitory activities with
the exception of compound 12p. When the substitution pattern was compared within this
This article is protected by copyright. All rights reserved.

series, the para-substitutents gave more potent analogs. Further evaluation revealed that
halogenated analogs were preferred at this position and compound 12l with para-iodine was
the most potent one with nanomolar potency. This may suggest the substituent size is
important at the para-position of the phenyl ring. To extend the confirmation of this notion,
compounds 12m-p were designed and evaluated. The results demonstrated that inhibitory
potency of 12m to HDACs was comparable to that of 12l and compounds with more bulky
substituents such as 12n-p showed decreased potency. Collectively, this may suggest that the
optimal size and at this position are needed to produce optimal interactions and consequently
inhibitory activities.
3.3 Molecular docking of 12m to HDAC2
In order to better understand the inhibition of 12m to HDACs, docking studies were
conducted employing structure of HDAC2. As shown in Fig 3, the linker of 12m stretches to
the bottom of the tunnel and the hydroxamic acid moiety orient can chelate properly with the
zinc ion, which is consistent with the chelating type of SAHA. Compared to the sole
hydrophobic interaction between the phenyl of SAHA and the amino acid residues around the
external motif, the p-methylphenyl and the indole ring of 12m can potentially double enhance
the ligand–receptor bindings of the surface plot near the opening of the tunnel. In addition,
the hydroxyl and carbonyl oxygens of 12m have four H-bond interactions with His145,
His146, Tyr308 and Asp104, while only two H-bond interactions of SAHA with His146 and
Asp104 were displayed. Moreover, the benzene ring, as part linker of 12m, has π-π stacking
interactions with the phenyl group of Phe155, which also improves its binding ability to the
active site of HDAC2. All the information observed above could rationalize the better
inhibitory activity of 12m than SAHA.
This article is protected by copyright. All rights reserved.

3.4 HDAC isoform selectivity of 12m
The studies employing the cellular extract demonstrated that compound 12m was the
most potent analog and slightly potent than SAHA under these experimental conditions. To
further ascertain HDAC isoform-selectivity of 12m, we conducted enzymatic inhibitory
assays against HDAC1 (Class I), HDAC8 (Class I), HDAC6 (Class IIb), HDAC4 (Class IIa),
and HDAC11 (Class IV). Compound 12m exhibited almost no inhibition to HDAC4 at the
concentration of 100 μM and displayed IC50 values close to the magnitude of micromole or
nanomole versus other HDAC isoforms, which demonstrated that 12m showed similar
overall selectivity profile with control PXD101.
3.5 In vitro antiproliferative assay
HDACs have been indicated significant roles in the proliferation and differentiation of
cancer cells21,22. In the following research, we conducted in vitro antiproliferative evaluation
of target compounds in cellular assays. Firstly, we tested the inhibition rate of all synthesized
compounds at 5 μM against two human leukemia cell lines (U937 and HEL) 23. As the
preliminary results showed in Fig 4, compounds 8d, 12d, 12j and 12m were selected as the
representative ones for further antiproliferative evaluation in vitro against five frequently
used cancer cell lines (Table 3) and exhibited comparable potency to SAHA in suppressing
the growth of HL60 cells, but less inhibitory activities aganist other cell lines than SAHA.
Interestingly, compound 8d with less potent inhibition in cellular extracts assay, while overall
displayed similar activities compared to SAHA in vitro antiproliferative assay. It was likely
8d interacted with complicated binding sites in cancer cells of which main ingredients
distinguished from the Hela cellular extracts.
This article is protected by copyright. All rights reserved.

Conclusions
In this article, a series of N1-hydroxyterephthalamide derivatives with indole cap analogs
were designed, synthesized and biologically characterized as HDACIs. Compounds 12k, 12l,
and 12m displayed potent HDAC inhibitory activity compared with control drug SAHA in
vitro HeLa cell extract inhibitory fluorescence assay. The most potent compound 12m was
also verified by molecular docking studies in the active site of HDAC2 and showed similar
overall selectivity profile with control PXD101 in HDAC isoform selectivity assay. Four
selected compounds (8d, 12d, 12j, 12m) demonstrated more potent or comparable
antiproliferative activities to SAHA in vitro antiproliferative assay. We found
antiproliferative activity of 12m was less potent than SAHA, which was inferior to their
enzyme inhibitory activity. One main reason could be that 12m was limited by its poor
transcellular permeability so that its specific binding with HDACs was weaker in cells. In
addition, 12m was likely to be disintegrated by other enzymes in cells before approaching
active site of HDACs. To summarise, the results revealed that N1-hydroxyterephthalamide
with an indole cap could serve as novel chemical scaffold to develop more potent HDACIs.
Acknowledgements
This work was supported by National Major Scientific and Technological Special Project
for ‘‘Significant New Drugs Development” of China (Grant no. 2012ZX09103101-015).
Conflict of interest
The authors declare no conflict of interest.
This article is protected by copyright. All rights reserved.

References and notes
1. Sharma, S.; Kelly T. K.; Jones, P. A., Epigenetics in cancer. Carcinogenesis 2010, 31(1), 27-36.
2. Parbin, S., Kar, S., Shilpi, A., Sengupta, D., Deb, M., Rath, S. K., & Patra, S. K., Histone Deacetylases A
Saga of Perturbed Acetylation Homeostasis in Cancer. Journal of Histochemistry & Cytochemistry 2014, 62(1),
11-33.
3. Dokmanovic, M.; Clarke, C.; Marks, P. A., Histone deacetylase inhibitors: overview and
perspectives. Molecular cancer research 2007, 5(10), 981-989.
4. López, J. E.; Sullivan, E. D.; Fierke, C. A., Metal-dependent deacetylases: Cancer and epigenetic
regulators. ACS chemical biology 2016, 11(3), 706-716.
5. Zhang L.; Han Y.; Jiang Q., et al. Trend of histone deacetylase inhibitors in cancer therapy: isoform
selectivity or multitargeted strategy. Medicinal research reviews 2015, 35(1), 63-84.
6. Grant, S.; Easley, C.; Kirkpatrick, P., Vorinostat. Nature reviews Drug discovery, 2007, 6(1), 21-22.
7. McGraw, A. L., Romidepsin for the treatment of T-cell lymphomas. American journal of health-system
pharmacy : AJHP : official journal of the American Society of Health-System Pharmacists 2013, 70 (13),
1115-22.
8. Chan, D.; Zheng, Y.; Tyner, J. W.; Chng, W. J.; Chien, W. W.; Gery, S.; Leong, G.; Braunstein, G. D.;
Koeffler, H. P., Belinostat and panobinostat (HDACI): in vitro and in vivo studies in thyroid cancer. Journal of
cancer research and clinical oncology 2013, 139 (9), 1507-14.
9. Gimsing, P., Belinostat: a new broad acting antineoplastic histone deacetylase inhibitor. Expert opinion on
investigational drugs 2009, 18 (4), 501-8.
10. Cheng, T.; Grasse, L.; Shah, J.; Chandra, J., Panobinostat, a pan-histone deacetylase inhibitor: rationale for
and application to treatment of multiple myeloma. Drugs of today 2015, 51 (8), 491-504.
11. Miller, T. A.; Witter, D. J.; Belvedere, S., Histone deacetylase inhibitors. Journal of medicinal chemistry
2003, 46 (24), 5097-116.
This article is protected by copyright. All rights reserved.

12. Olson, D. E.; Wagner, F. F.; Kaya, T.; Gale, J. P.; Aidoud, N.; Davoine, E. L.; Lazzaro, F.; Weiwer, M.;
Zhang, Y. L.; Holson, E. B., Discovery of the first histone deacetylase 6/8 dual inhibitors. Journal of medicinal
chemistry 2013, 56 (11), 4816-20.
13. DeSimone, R. W.; Currie, K. S.; Mitchell, S. A.; Darrow, J. W.; Pippin, D. A., Privileged structures:
applications in drug discovery. Combinatorial chemistry & high throughput screening 2004, 7 (5), 473-94.
14. Costantino, L.; Barlocco, D., Privileged structures as leads in medicinal chemistry. Current medicinal
chemistry 2006, 13 (1), 65-85.
15. Venugopal, B.; Baird, R.; Kristeleit, R. S.; Plummer, R.; Cowan, R.; Stewart, A.; Fourneau, N.; Hellemans,
P.; Elsayed, Y.; McClue, S.; Smit, J. W.; Forslund, A.; Phelps, C.; Camm, J.; Evans, T. R.; de Bono, J. S.; Banerji,
U., A phase I study of quisinostat (JNJ-26481585), an oral hydroxamate histone deacetylase inhibitor with
evidence of target modulation and antitumor activity, in patients with advanced solid tumors. Clinical cancer
research : an official journal of the American Association for Cancer Research 2013, 19 (15), 4262-72.
16. Arts, J.; King, P.; Marien, A.; Floren, W.; Belien, A.; Janssen, L.; Pilatte, I.; Roux, B.; Decrane, L.; Gilissen,
R.; Hickson, I.; Vreys, V.; Cox, E.; Bol, K.; Talloen, W.; Goris, I.; Andries, L.; Du Jardin, M.; Janicot, M.; Page,
M.; van Emelen, K.; Angibaud, P., JNJ-26481585, a novel "second-generation" oral histone deacetylase inhibitor,
shows broad-spectrum preclinical antitumoral activity. Clinical cancer research : an official journal of the
American Association for Cancer Research 2009, 15 (22), 6841-51.
17. Zhang, Y.; Yang, P.; Chou, C. J.; Liu, C.; Wang, X.; Xu, W., Development of -Hydroxycinnamamide-Based
Histone Deacetylase Inhibitors with Indole-Containing Cap Group. ACS medicinal chemistry letters 2013, 4 (2),
235-238.
18. Zhang Y.; Feng J.; Liu C.; Zhang L.; Jiao J.; Fang H.; Su L.; Zhang X.; Zhang J.; Li M.; Wang B.; Xu W.,
Design, synthesis and preliminary activity assay of 1, 2, 3, 4-tetrahydroisoquinoline-3- acid derivatives as novel
Histone deacetylases (HDACs) inhibitors. Bioorganic & medicinal chemistry 2010, 18(5), 1761-1772.
19. Evans, M. J.; Morris, G. M.; Wu, J.; Olson, A. J.; Sorensen, E. J.; Cravatt, B. F., Mechanistic and structural
requirements for active site labeling of phosphoglycerate mutase by spiroepoxides. Molecular bioSystems 2007, 3
(7), 495-506.
This article is protected by copyright. All rights reserved.

20. Li, X.; Wu, J.; Li, X.; Mu, W.; Liu, X.; Jin, Y.; Xu, W.; Zhang, Y., Development of N-hydroxybenzamide
derivatives with indole-containing cap group as histone deacetylases inhibitors. Bioorganic & medicinal
chemistry 2015, 23 (19), 6258-70.
21. Bolden, J. E.; Peart, M. J.; Johnstone, R. W., Anticancer activities of histone deacetylase inhibitors. Nature
reviews. Drug discovery 2006, 5 (9), 769-84.
22. Xu, W. S.; Parmigiani, R. B.; Marks, P. A., Histone deacetylase inhibitors: molecular mechanisms of action.
Oncogene 2007, 26 (37), 5541-52.
23. Li X.; Inks E. S.; Li X.; Hou J.; Chou C. J.; Zhang J.; Jiang Y.; Zhang Y.; Xu W., Discovery of the first
N-hydroxycinnamamide-based histone deacetylase 1/3 dual inhibitors with potent oral antitumor activity. Journal
of medicinal chemistry 2014, 57(8): 3324-3341.
Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure 1. Pharmacophore model and HDACIs approved by FDA
Figure 2. Design of novel series of HDACIs
Figure 3. The docking modes of compounds 12m (yellow molecular) and SAHA (green
molecular) in the active site of HDAC2, derived by modification of PDB code 4LXZ with
Tripos sybyl x 2.0 (Zinc ion is indicated green in the bottom of the tunnel)
Figure 4. Inhibition rate of synthesized compounds to U937 and HEL at 5 μM
Scheme 1. The synthetic route for target compounds 8a-h and 12a-p.
Table 1. The chemical structures and HDACs inhibitory activities of
N1-hydroxyterephthalamide Derivatives
Table 2. Inhibitory selectivity toward HDAC of compound 12m.
Table 3. In vitro antiproliferative activity of 8d, 12d, 12j and 12m.
Data S1. Physical properties and spectrum data of target compounds.
This article is protected by copyright. All rights reserved.

Tables
Table 1. The chemical structures and HDACs inhibitory activities of
N1-hydroxyterephthalamide Derivatives
IC50a of Hela
IC50a of Hela
Compd
8a
R
Compd
12f
R
extract(μM)
extract(μM)
0.227
0.360
0.189
0.435
0.202
0.150
0.096
0.078
0.074
0.413
0.335
1.654
1.742
＞ 25
8b
12g
12h
12i
8c
8d
0.527
0.504
0.683
0.416
0.361
0.346
8e
12j
8f
12k
12l
8g
8h
12m
12n
12o
12a
12b
This article is protected by copyright. All rights reserved.

0.301
1.287
0.131
12c
12p
0.225
0.234
12d
12e
SAHA
aAssays were performed in replicate (n≥2); The SD values are < 20% of the mean.
Table 2. Inhibitory selectivity toward HDAC of compound 12m.
IC50a(μM)
compd
Class I
HDAC1
Class IIa
HDAC4
>100
Class IIb
HDAC6
0.168
Class IV
HDAC11
3.50
HDAC8
5.50
0.219
0.034
12m
0.353
9.9
0.027
25
PXD-101
aAssays were performed in replicate (n≥2); the SD values are <20% of the mean.
Table 3. In vitro antiproliferative activity of 8d, 12d, 12j and 12m.
a
IC50 (μM)
compd
K562
U266
KG-1
HL60
MCF-7
6.75 0.96
10.51 0.96
2.63 0.78
6.27 0.46
8.72 0.06
6.56 0.13
3.25 0.61
12.59 1.7
7.07 0.36
8d
12d
6.25 0.07 17.38 1.57 10.23 0.63
7.68 0.53
5.71 0.20
1.76 0.12
15.64 0.64
10.09 0.41
1.98 0.21
8.05 1.17
9.09 1.39
1.23 0.14
12j
10.80 0.33 13.33 0.38
9.37 0.39 1.63 0.29
12m
SAHA
aAssays were performed in replicate (n≥2); the SD values are <20% of the mean.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.