2-Benzazolyl-4-Piperazin-1- Ylsulfonylbenzenecarbohydroxamic acids as novel selective histone deacetylase-6 inhibitors with antiproliferative activity

Article abstract of DOI:10.1371/journal.pone.0134556

We have screened our compound collection in an established cell based assay that measures the derepression of an epigenetically silenced transgene, the locus derepression assay. The screen led to the identification of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1- yl]sulfonylbenzenecarbohydroxamic acid (9b) as an active which was found to inhibit HDAC1. In initial structure activity relationships study, the 1-methylbenzimidazole ring was replaced by the isosteric heterocycles benzimidazole, benzoxazole, and benzothiazole and the position of the hydroxamic acid substituent on the phenyl ring was varied. Whereas compounds bearing a para substituted hydroxamic acid (9a-d) were active HDAC inhibitors, the meta substituted analogues (8a-d) were appreciably inactive. Compounds 9a-d selectively inhibited HDAC6 (IC50 = 0.1-1.0μM) over HDAC1 (IC50 = 0.9-6μM) and moreover, also selectively inhibited the growth of lung cancer cells vs. patient matched normal cells. The compounds induce a cell cycle arrest in the S-phase while induction of apoptosis is neglible as compared to controls. Molecular modeling studies uncovered that the MMGBSA energy for interaction of 9a-d with HDAC6 was higher than for HDAC1 providing structural rationale for the HDAC6 selectivity.

Full text of DOI:10.1371/journal.pone.0134556

RESEARCH ARTICLE
2-Benzazolyl-4-Piperazin-1-
Ylsulfonylbenzenecarbohydroxamic Acids as
Novel Selective Histone Deacetylase-6
Inhibitors with Antiproliferative Activity
Lei Wang1, Marina Kofler2, Gerald Brosch3, Jelena Melesina4, Wolfgang Sippl4, Elisabeth
D. Martinez1,5*, Johnny Easmon2*
1 Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, TX, United
States of America, 2 Institute of Pharmacy, Department of Pharmaceutical Chemistry, Center for Chemistry
and Biomedicine, University of Innsbruck, Innrain 80–82, A-6020 Innsbruck, Austria, 3 Division of Molecular
Biology, Center for Chemistry and Biomedicine, Innsbruck Medical University Innrain 80–82, A-6020
Innsbruck Austria, 4 Institute of Pharmacy, Department of Medicinal Chemistry, Martin-Luther-Universität
Halle-Wittenberg, D-06099 Halle/Saale, Germany, 5 Department of Pharmacology, UT Southwestern
Medical center, Dallas, TX, United States of America
* johnny.easmon@uibk.ac.at (JE); elisabeth.martinez@utsouthwestern.edu (EM)
OPEN ACCESS
Citation: Wang L, Kofler M, Brosch G, Melesina J,
Sippl W, Martinez ED, et al. (2015) 2-Benzazolyl-4-
Piperazin-1-Ylsulfonylbenzenecarbohydroxamic
Acids as Novel Selective Histone Deacetylase-6
Inhibitors with Antiproliferative Activity. PLoS ONE 10
(12): e0134556. doi:10.1371/journal.pone.0134556
Abstract
We have screened our compound collection in an established cell based assay that mea-
sures the derepression of an epigenetically silenced transgene, the locus derepression
assay. The screen led to the identification of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-
yl]sulfonylbenzenecarbohydroxamic acid (9b) as an active which was found to inhibit
HDAC1. In initial structure activity relationships study, the 1-methylbenzimidazole ring was
replaced by the isosteric heterocycles benzimidazole, benzoxazole, and benzothiazole and
the position of the hydroxamic acid substituent on the phenyl ring was varied. Whereas
compounds bearing a para substituted hydroxamic acid (9a-d) were active HDAC inhibitors,
the meta substituted analogues (8a-d) were appreciably inactive. Compounds 9a-d selec-
tively inhibited HDAC6 (IC50 = 0.1–1.0μM) over HDAC1 (IC50 = 0.9–6μM) and moreover,
also selectively inhibited the growth of lung cancer cells vs. patient matched normal cells.
The compounds induce a cell cycle arrest in the S-phase while induction of apoptosis is
neglible as compared to controls. Molecular modeling studies uncovered that the MM-
GBSA energy for interaction of 9a-d with HDAC6 was higher than for HDAC1 providing
structural rationale for the HDAC6 selectivity.
Editor: Marta M. Alonso, University Hospital of
Navarra, SPAIN
Received: August 11, 2014
Accepted: July 13, 2015
Published: December 23, 2015
Copyright: © 2015 Wang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This project was partly funded by the NIH
(R01CA12526901 to E.D.M., 1R21AI11622201 to I.
D./E.D.M. P50-CA70907 to J.D.M., subaward to E.D.
M.) by the Doctor’s Cancer Foundation (Nolan Miller
Lung Cancer grant to E.D.M.), by the Friends of the
Cancer Center, by the Welch Foundation (grants to E.
D.M.), by the National Cancer Institut
Introduction
Aberrant regulation of gene expression is at the basis of several human diseases including
many forms of cancer. This is partly the result of deregulation of DNA methylation or the
R01CA12526901 to E.D.M., and by CPRIT
(RP120717). The funders had no role in study design, post translational modification of histone tails. The cellular enzymes that mediate these
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Novel HDAC6 Inhibitors
data collection and analysis, decision to publish, or
preparation of the manuscript.
modifications include DNA-methyltranferases (DNMTs), the recently described TET proteins
that hydroxylate methylated DNA, histone acetyl transferases (HATs), histone deacetylases
(HDACs), histone methyltransferases (HMTs), histone demethylases (HDMs) and histone
kinases. Together, these enzymes influence transcription by maintaining tissue–specific epige-
netic and transcriptional patterns as well as by acting as co-regulators for transcription factors.
In disease, this regulation of the transcriptional process can be altered as a consequence of
changes in the expression or function of epigenetic enzymes [1,2]. The most pharmacologically
targeted in this class have been the HDACs.
Competing Interests: The authors have declared
that no competing interest exist.
HDACs influence the level of histone acetylation by removing the terminal ε-acetyl function
of lysines. This activity condenses the chromatin into a closed structure resulting in the repres-
sion of transcription whereas the acetylation of histone lysine by HATs leads to an open chro-
matin structure thus allowing transcriptional initiation and gene expression. Currently there
are 18 known human HDAC isoforms that are commonly divided into 4 classes, namely Class
I (HDACs 1, 2, 3, and 8), Class II (HDACs, 4, 5, 6, 7, 9, and 10), Class III (SIRT 1–7) and class
IV (HDAC 11). Whereas Class I, II and IV are zinc dependent, the class III enzymes require
NAD+ for their activity. Most inhibitors of Class I, II and IV HDACs thus act by chelating
active site zinc [3].
Since it was discovered that the antiproliferative activity of trichostatin A (TSA) was medi-
ated through the inhibition of HDACs, [4] much effort has been invested in finding new inhib-
itors of this enzyme family. A diverse group of compounds discovered to date fall into four
chemotypes: namely, hydroxamic acids, short fatty acids, cyclic tertrapeptides and benzamides
of which several are currently in clinical development [5,6]. Of this first generation com-
pounds, SAHA (Vorinostat), PXD101 (Belinostat), Romidepsin (Istodax), and RAS2410
(Resminostat) have received approval for the treatment of various cancers and were thought to
target several or most members of the HDAC family. Although during the pre-clinical investi-
gations these pan-HDAC inhibitors were deemed safe, in the clinical setting these compounds
have exhibited various toxicities. For this reason there is an ongoing discussion of whether iso-
form-selective HDAC inhibitors would be more advantageous than pan-inhibitors [7]. Recent
studies of the various isoform enzymes have revealed subtle differences in the amino acid make
up of the active site which can be exploited in the development of isoform or class selective
compounds [8]. In fact, this approach has been successful in achieving several class or isoform
selective inhibitors, for example MS-275 (HDAC1), MGCD0103 (HDAC1,2), Tubacin and
Tubastatin A (HDAC6), and PCI-34051 and SB-379278A (HDAC8) [9]. Furthermore, using
molecular modeling and biochemical studies Dominguez and co-workers recently described a
subset of small molecule hydroxamates as class IIa (HDAC4,5,7,9) specific inhibitors [10].
With the goal of increasing the diversity and selectivity of epigenetic modulators, we have
screened our compound collection (Department of Pharmaceutical Chemistry, University of
Innsbruck) against an established cell based assay that measures the derepression of an epige-
netically silenced GFP transgene, the LDR or locus derepression assay [11,12]. This assay is
sensitive to known HDAC inhibitors including TSA, depsipeptide and butyrate as well as to
other epigenetic modulators such as 5-azadeoxycytidine, which activate the expression of the
GFP transgene. The screen led to the identification of several active compounds with diverse
molecular structures. One of the compounds, JIB-04, a pyridine hydrazone derivative modu-
lates Jumonji histone demethylase activity and selectively inhibits cancer growth in vivo [13].
Among the active compounds was one which bears a hydroxamic acid functionality, (4-[4-
(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydroxamic acid, (9b), that
was originally synthesized for studies of potential 11ß-hydroxysteroid dehydrogenase 1 inhibi-
tors [14]. Here, we describe the activity of 9b and the synthesis and characterization of 9b ana-
logs. We find that para-substituted hydroxamic acid analogs of 9b inhibit HDAC activity
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Novel HDAC6 Inhibitors
showing preference for HDAC6 over HDAC1 and have cancer selective antiproliferative prop-
erties. In addition to inhibition of HDAC activity, the compounds also induce a cell cycle arrest
in cancer cells decreasing the cell number.
Materials and Methods
Chemistry
The synthesis of the mono-substituted 2-benzazolpiperazines 5a,b,d has been described in the
literature by reacting 2-chlorobenzazols 1a,b,d with excess piperazine hydrate (4) [15,16].
Although it was reported that compounds 5a,b,d were obtained in high yields (70–90%), in
our hands the major isolated product was the 1,4-diarylated piperazine in 80% yield. Com-
pounds 5a,b,d, were then prepared in a 2-step reaction utilizing a mono-N-protected pipera-
zine derivative (2) as described by Shafic et al (Fig 1) [17].
Ethylcarboxylate piperazine derivatives 3a,b,d were obtained from 2-chlorobenzazoles 1a,b,
d by nucleophilic substitution of the chlorine atom with ethyl N-piperazinecarboxylate 2. Sub-
sequent removal of the ethylcarboxylate moiety with aqu. 40% HBr at 70°C gave the benzazol-
2-yl piperazines 5a,b,d, in good yields. 2-Piperazin-1-yl-benzoxazole 5c was synthesized by the
treatment of 2-chlorobenzoxazole 1c with piperazine hydrate 4 at 0°C in CH2Cl2 [18].
The sulfonamide derivatives 6a-d and 7a-d became accessible by reacting compounds 5a-d
with either 3-chlorosulfonylbenzoic acid or 4-chlorosulfonylbenzoic acid, respectively. We
adapted the mild and efficient one step conversion of carboxylic acids to hydroxamic acids
reported by Reddy et al [19] for the synthesis of the target compounds 8a-d and 9a-d.
Fig 1. Synthesis of target compounds. Reagents; i) 120°C, 3 h.; ii) 40% aqu. HBr, 70°C, 12 h; iii) Dry
CH2Cl2, N(CH2CH3)3, 0°C; iv) 3-chlorosulfonylbenzoic acid or 4-chlorosulfonylbenzoic acid, N(CH2CH3)3,
THF, 0°C to RT; v) (a) N-methyl morpholine, ethyl chloroformate, THF, 0°C to RT, (b) hydroxylamine HCl,
KOH, EtOH, 0°C to RT.
doi:10.1371/journal.pone.0134556.g001
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Novel HDAC6 Inhibitors
Carboxylic acids 6/7a-d were first transformed to the acid anhydride by treatment with ethyl
chloroformate at 0°C utilizing N-methyl morpholine as a base. Then freshly prepared hydrox-
ylamine solution obtained by reacting hydroxylamine hydrochloride with potassium hydroxide
in methanol was added to the acid anhydride mixture to obtain compounds 8a-d and 9a-d in
high yields (Fig 1).
General Information. Infrared spectra (IR) were recorded from KBr pellets on a Mattson
Galaxy Series FTIR 3000 spectrophotometer. 1H NMR spectra were recorded from DMSO-d6
solutions on a Varian Gemini 2000 (1H: 199.98 MHz) spectrometer. The centre of the solvent
signal was used as internal standard, which was related to TMS with δ 2.49 ppm (1H). Assign-
ments are based on chemical shift considerations. Melting points were determined on a Reich-
ert Thermovar hot stage microscope and are uncorrected. Elemental analyses were performed
at the "Institut für Physikalische Chemie", University of Vienna, Austria and the data for
C, H, N are within 0.4% of the calculated values. Reactions were monitored by TLC using
Polygram SIL G/UV254 (Macherey-Nagel) plastic backed plates (0.25 mm layer thickness)
and visualized using a UV lamp. Column chromatography was performed using Kieselgel
60 (0.040–0.063 mm).
2-Chlorobenzimidazole, 2-chlorobenzoxazole, 2-chlorobenzothiazole, 3-chlorosulfonyl
benzoic acid and 4-chlorosulfonyl benzoic acid were purchased from Sigma-Aldrich Co.
Abbreviations: CH2Cl2 (dichloromethane), DIPE (diisopropyl ether), EA (ethylacetate), EtOH
(ethanol), MeOH (methanol), 2-PrOH (2-propanol), and THF (tetrahydrofuran).
Chemical Synthesis of Compounds
Synthesis of 2-Chloro-1-methyl-benzimidazole (1b): 2-Chlorobenzimidazole (10 g, 65.56 mmol)
was dissolved in 80 ml of 2.5N NaOH (175 mM) and dimethyl sulfate (11.0 mL, 116 mM) was
added drop wise under stirring at room temperature. After the addition the mixture was stirred
for further 2 h and the precipitate formed was filtered by suction and the product washed sev-
eral times with ice-water mixture till the filtered solution was neutral. Petrol ether was sucked
through the solid product several times and then dried in vacuo to afford a light brown solid
(8.76 g, 81% yield). 1H NMR (200 MHz, d6-DMSO, δ): 3.78 (s, 3H, CH3), 7.26 (dq, J = 1.2Hz,
J = 7.4Hz, BZI-H5/H6), 7.54–7.60 (m, 2H, BZI-H4/H7).
General method for synthesis of compounds 3a,b,d: A mixture of 2-chlorobenzazoles 1a, 1b,
or 1d (1 equivalent) and ethoxycarbonylpiperazine 2 (3 equivalents) was heated at 120°C
under stirring and the reaction followed by TLC (CH2Cl2/EA 7:3). After completion the reac-
tion mixture was allowed to cool to room temperature and the mixture dissolved in 200 mL
CH2Cl2. The organic phase was extracted with saturated NaHCO3 solution (3 x 100 mL), then
with dist. H2O (2 x 50 mL), sat. NaCl solution (100 mL) and dried over anhy. Na2SO4. After fil-
tration, the organic phase was evaporated to dryness. The precipitate formed after treatment of
the residue with DIPE and stirring in the cold (ice/water mixture) was filtered and dried.
Ethyl 4-(1H-benzimidazol-2-yl)-piperazin-1-carboxylate (3a): 2-Chlorobenzimidazole (1.16
g, 5.88 mmol) and ethoxycarbonylpiperazine (2.85 g, 18.04 mmol) with a reaction time of 1h to
yield a white product (1.06 g, 97%). 1H NMR (200 MHz, d6-DMSO, d): 1.20 (t, J = 6.7Hz, 3H,
CH3), 3.49–3.58 (m, 8H, 8x Pip-H), 4.07 (q, J = 6.7Hz, 2H, CH2), 6.39 (m, 2H, BZI-H5/6), 7.17
(m, 2H, BZI-H4/7), 11.39 (s, 1H, NH).
Ethyl 4-(1-methyl-benzimidazol-2-yl)-piperazin-1-carboxylate (3b): 2-Chloro-1-methylben-
zimidazole (2.00 g, 12.04 mmol) and ethoxycarbonylpiperazine (3.80 g, 24.05 mmol) heated at
125°C for 1h to yield a light yellow product (2.80 g, 81%). 1H NMR (200 MHz, d6-DMSO, d):
1.21 (t, J = 7.0Hz, 3H, CH3), 3.19–3.31 (m, 4H, 4x Pip-H), 3.55–3.62 (m, 7H, 4x Pip-H,
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Novel HDAC6 Inhibitors
N-CH3), 4.08 (q, J = 7,2Hz, 2H, CH2), 7.05–7.15 (m, 2H, BZI-H5/6), 7.31–7.43 (m, 2H, Bzi-
H4/7).
Ethyl 4-(benzothiazol-2-yl)-piperazin-1-carboxylate (3d): 2-Chlorobenzimidazole (1.16 g,
6.82 mmol) and ethoxycarbonylpiperazine (3.24 g, 20.50 mmol) heated together for 5 h to
yield a light yellow product (1.85 g, 93%). 1H NMR (200 MHz, d6-DMSO, d): 1.20 (t, J = 7.0Hz,
3H, CH3), 3.40–3.65 (m, 8H, 8x Pip-H), 4.07 (q, J = 7.0Hz, 2H, CH2), 7.03–7.12 (m, 1H,
BZT-H6), 7.24–7.32 (m, 1H, BZT-H5), 7.45–7.48 (m, 1H, BZT-H4), 7.75–7.79 (m, 1H,
BZT-H7).
General method for synthesis of compounds 5a,b,d: Compound 5a, 5b, or 5d was treated
with 48% HBr in water and the mixture heated at 70°C under stirring for 24 h. The reaction
mixture was allowed to cool to room temperature and evaporated to dryness. The residue was
dissolved in water (100 mL) and made basic with conc. NH3 and extracted with CH2Cl2
(4 x 100 mL). The combined organic phase was washed with dist. water (2 x 50 mL), sat. NaCl
solution, dried over anhyd. Na2SO4, filtered and the solvent evaporated to dryness. The prod-
uct was recrystallised from DIPE.
2-(Piperazin-1-yl)benzimidazole (5a): Heating ethyl 4-(1H-benzimidazol-2-yl)-piperazin-
1-carboxylate 6a (2.00 g, 7.29 mmol) and 48% HBr (40 mL) yielded a white product (1.37 g,
93%). 1H NMR (200 MHz, d6-DMSO, d): 3.01–3.60 (m, 4H, 4x Pip-H), 3.89–3.92 (m, 4H,
4x Pip-H), 7.27–7.33 (m, 2H, BZI-H5/6), 7.42–7.49 (m, 2H, BZI-H4/7), 9.18 (s, 1H, NH).
1-Methyl-2-(piperazin-1-yl)benzimidazole (5b): Ethyl 4-(1-methyl-benzimidazol-2-yl)-
piperazin-1-carboxylate 6b (2.80 g, 9.71 mM) and 48% HBr (40.5 mL) to yield a light yellow
product (1.93 g, 92%). 1H NMR (200 MHz, d6-DMSO, d): 2.5–2.90 (m, 4H, 4x Pip-H),
3.11–3.21 (m, 4H, 4x Pip-H), 3.58 (s, 3H, N-CH3), 7.05–7.12 (m, 2H, BZI-H5/H6), 7.29–7.41
(m, 2H, BZI-H4/H7).
2-(Piperazin-1-yl)benzothiazole (5d): Ethyl 4-(benzothiazol-2-yl)-piperazin-1-carboxylate
(6c) (1.50 g, 5.15 mM) and 48% HBr (24.5 mL) to yield a colourless product (1.08 g, 96%).
1H NMR (200 MHz, d6-DMSO, d): 2.76 (m, 4H, 4x Pip-H), 3.43–3.48 (m, 4H, 4x Pip-H),
6.99–7.11 (m, 1H, BZT-H6), 7.21–7.31 (m, 1H, BZT-H5), 7.41–7.49 (m, 1H, BZT-H4),
7.71–7.75 (m, 1H, BZT-H7).
Synthesis of 2-(Piperazin-1-yl)benzoxazole (5c): 5.6g (65 mM) Piperazine (water free) and
3.27g (0.325 mol) triethylamine was dissolved in 50 ml of dry CH2Cl2 and the solution cooled
to 0°C in an ice/salt bath. 2-Chlorobenzoxazole (1c) dissolved in 20 mL of dry CH2Cl2 was
added dropwise to the solution under stirring keeping the solution at 0°C. After the addition,
the mixture was stirred for a further 30 min at 0°C and then quenched with 200 mL of ice-
water. After separation of the organic phase, the water phase was extracted with ethyl acetate
(2 x 100 mL), the combined organic phases was washed with sat. NaCl solution, dried over
anhyd. Na2SO4, filtered and evaporated to dryness. Compound 8d was isolated as a white solid
over column chromatography using silica gel with CH2Cl2/MeOH (5:1) as eluent. Yield 4.96 g
(75%). 1H NMR (200 MHz, d6-DMSO, d): 3.48–3.53 (m, 4H, 4x Pip-H); 3.17 (s, 1H, NH);
2.75–2.80 (m, 4H, 4x Pip-H); 6.99 (dt, J = 1.6Hz, J = 8Hz, 1H, BZO-H5); 7.18 (dt, J = 1.2Hz,
J = 7.6Hz, 1H, BZO-H6); 7.27 (dd, J = 1.4Hz, J = 7.6Hz, 1H, BZO-H7); 7.37 (dd, J = 0.6Hz,
J = 7.8Hz, 1H, BZO-H4).
General synthesis of the carboxylic acid derivatives 6a-d and 7a-d: 2-(Piperazin-1-yl)benza-
zolyl derivatives 5a-d (1 equivalent) and triethylamine (1.5 equivalents) were dissolved in
25 mL dry CH2Cl2 and cooled to 0°C in an ice salt bath. Then the required 3 or 4-Chloro-sulfo-
nylbenzoic acid (1.1 equivalents) dissolved in 15 mL dry THF was added dropwise. After the
addition, the reaction mixture was stirred at 0°C for a further 1 h and then allowed to warm to
room temperature and the course of the reaction followed by TLC (CH2Cl2/MeOH/konz. NH3:
898/100/2). After indicating the end of the reaction, the mixture was evaporated to dryness, the
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Novel HDAC6 Inhibitors
residue was dissolved in dist. water and made slightly acidic (pH 6) with glacial acetic acid and
stirred for about 30 mins. The solid formed was filtered by suction, washed with water and
then with petrol ether. The product was recrystallized from the appropriate solvent; the precip-
itate formed was filtered under suction and dried at 60°C under high vacuum.
3-[4(1H-Benzimidazol-2-yl)-piperazin-1-yl]sulfonyl-benzoic acid (6a): 2-(Piperazin-1-yl)
benzimidazole hydrobromide 5a (0.200 g. 1.06 mM), triethylamine (0.285 g, 4.22 mM) and
3-chlorosulfonylbenzoic acid (0.186 g, 1.27 mM) was reacted for overnight at RT to give col-
ourless crystals (0.295 g, 72%). Compound 6a was recrystalissed from EtOH (m. pt. 335°C,
decomposed). IR (KBr, v cm-1): 2994 (-OH), 1648 (C = O), 1613, 1560 (C = C, C = N), 1344
(-SO2-). 1H NMR (200 MHz, d6-DMSO, d): 2.98–3.12 (M, 4H, 4x PIP-H), 3.55–3.66 (m, 4H,
4x PIP-H), 6.87–6.92 (m, 2H, BZI-H5,6), 7.13–7.18 (m, 2H, BZI-H4,7), 7.73–781 (m, 1H,
Ar-H5), 7.98–8.02 (m, 1H, Ar-H6), 8.21–8.24 (m, 1H, Ar-H2,4).
3-[4-(1-Methylbenzimidazol-2-yl)piperazin-1-sulfonyl]-benzoic acid (6b): 1-Methyl-2-
(piperazin-1-yl)benzimidazole 5b (0.300 g. 1.38 mM), triethylamine (0.281 g, 2.76 mM) and
3-chlorosulfonylbenzoic acid (0.367 g, 1.67 mM) was reacted for 23 h at RT to give colourless
crystals (0.523 g, 93%). Compound 6b was recrystallised from EtOH (m. pt. 291–293°C). IR
(KBr, v cm-1): 2855 (-OH), 1714 (C = O), 1616, 1637 (C = C, C = N), 1349 (-SO2-). 1H NMR
(200 MHz, d6-DMSO, d): 3.14 (br s, 4H, 4x Pip-H), 3.33 (br. s, 4H, 4x Pip-H), 3.51 (s, 3H,
N-CH3), 7.05–7.09 (m, 2H, BZI-H6,5), 7.29–7.39 (m, 2H, BZI-H4,7), 7.79–7.86, (m, 1H,
Ph-H5), 8.02–8.06 (m, 1H, Ph-H6), 8.24–8.29 (m, 2H, Ph-H2,4).
3-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (6c): 2-(Piperazin-1-yl)benzoxazole
5c (0.300 g, 1.48 mM), triethylamine (0.299 g, 2.96 mM) and 3-chlorosulfonylbenzoic acid
(0.391 g, 0.18 mM) was reacted for 23 h at RT to give colourless crystals (0.545 g, 95%). Com-
pound 6c was recrystallized from EtOH (m. pt. 290–293°C, decomposses IR (KBr, v cm-1):
2867 (-OH), 1685 (C = O), 1640, 1581 (C = C, C = N), 1348 (-SO2-). 1H NMR (200 MHz,
d6-DMSO, d): 3.00–3.11 (m, 4H, 4x Pip-H), 3.69 (br s, 4H, 4x Pip-H), 6.95–7.03 (m, 1H,
BZO-H5 or 6), 7.08–7.15 (m, 1H, BZO-H5 or 6), 7.23–7.27 (m, 1H, BZO-H4), 7.34–7.37 (m, 1H,
BZO-H7), 7.73–7.8 (m, 1H, Ar-H5), 7.95–7.99 (m, 1H, Ar-H6), 8.19–8.23 (m, 1H, Ar-H2,4).
3-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (6d): 2-(Piperazin-1-yl)benzothia-
zole 5d (0.300 g. 1.37 mM), triethylamine (0.277 g, 2.73 mM) and 3-chlorosulfonylbenzoic acid
(0.362 g, 1.64 mM) was reacted overnight at RT to give colourless crystals (0.515 g, 93%). Com-
pound 6d was recrystallized from EtOH (m. pt. 375.5°C, decomposes). IR (KBr, v cm-1): 3441
(OH), 1704 (C = O), 1592, 1536 (C = N, C = C), 1350 (-SO2-). 1H NMR (200 MHz, d6-DMSO,
d): 3.06 (br. s., 4H, 4x Pip-H), 3.66 (br. s., 4H, 4x Pip-H), 7.01–7.09 (m, 1H, BZT-H6), 7.21–
7.29 (m, 1H, BZT-H5), 7.43 (d, J = 7.8Hz, BZT-H4), 7.65–7.75 (m, 2H, 2x Ar-H5,6), 7.87 (d,
J = 7.6 Hz, 1H, BZT-H7), 8.18–8.22 (m, 2H, 2x Ar-H2,4).
4-[4(1H-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-benzoic acid (7a): 2-(Piperazin-1-yl)benz-
imidazole hydrobromide 5a (0.400 g, 1.06 mM), triethylamine (0.570 g, 4.22 mM) and 4-chlor-
osulfonylbenzoic acid (0.373 g, 1.27 mM) was reacted overnight at RT to give colourless
crystals (0.498 g, 92%). Compound 7a was recrystallized from EtOH (m. pt. > 350°C). IR
(KBr, v cm-1): 2894 (-OH), 1638 (C = O), 1610 (C = N, C = C). 1H NMR (200 MHz, d6-DMSO,
d): 2.98–3.12 (m, 4H, 4x Pip-4H), 3.55–3.66 (m, 4H, 4x Pip-4H), 6.87–6.92 (m, 2H, BZI-H5,6),
7.11–7.16 (m, 2H, BZI-H4,7), 7.87 (ABd, J = 8.0 Hz, 2H, ArH-2,6), 8.52 (ABd, J = 8.2 Hz, 2H,
ArH-3,5).
4-[4-(1-Methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzoic acid (7b): 1-Methyl-2-
(piperazin-1-yl)benzimidazole 5b (0.300 g. 1.38 mM), triethylamine (0.281 g, 2.76 mM) and
4-chlorosulfonylbenzoic acid (0.367 g, 1.67 mM) was reacted for 3 days at RT to give colourless
crystals (0.550 g, 99%). Compound 7b was recrystallised from EtOH (m. pt. 340°C, decomposi-
tion). IR (KBr, v cm-1): 2867 (-OH), 1704 (C = O), 1614, 1586 (C = C, C = N), 1354 (-SO2-),
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Novel HDAC6 Inhibitors
1H NMR (200 MHz, d6-DMSO, d): 3.14 (br.s, 4H, 4x Pip-H), 3.32 (br.s, 4H, 4x Pip-H), 3.51 (s,
3H, N-CH3), 7.04–7.09 (m, 2H, BZI-H5,6), 7.29–7.39 (m, 2H, BZI-H4,7), 7.90 (ABd, J = 8.4Hz,
2H, Ph-2,6), 8.19 (ABd, J = 8.4Hz, 2H, Ph-3,5).
4-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (7c): 2-(Piperazin-1-yl)benzoxazole
5c (0.200 g. 1.48 mM), triethylamine (0.199 g, 2.96 mM) and 4-chlorosulfonylbenzoic acid
(0.260 g, 1.78 mM) was reacted overnight at RT to give colourless crystals (0.329 g, 85%). Com-
pound 7c was recrystallized from EtOH (m. pt. > 350°C). IR (KBr, v cm-1): 2917 (-OH), 1689
(C = O), 1582 (C = C, C = N), 1346 (-SO2-). 1H NMR (200 MHz, d6-DMSO, d): 3.09 (br s, 4H,
4x Pip-4H), 3.69 (br s, 4H, 4x Pip-4H), 6.96–7.03 (m, 1H, BZO-H5 or 6), 7.08–7.16 (m, 1H,
BZO-H5 or 6), 7.25 (d, J = 8.0Hz, 1H, BZO-H7), 7.36 (d, J = 7.6 Hz, 1H, BZO-H4), 7.86 (ABd,
J = 7.8 Hz, 2H, ArH2,6), 8.14 (ABd, J = 7.8Hz, 2H, ArH3,5).
4-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-benzoic acid (7d): 2-(Piperazin-1-yl)benzothia-
zole 5d (0.300 g. 1.38 mM), triethylamine (0.277 g, 2.76 mM) and 4-chlorosulfonylbenzoic acid
(0.362 g, 1.56 mM) was reacted for 23 h at RT to give colourless crystals (0.552 g, 98%). Com-
pound 7d was recrystallized from EtOH (m. pt. 325–327°C). IR (KBr, v cm-1): 2992 (-OH),
1689 (C = O), 1593 (C = C, C = N), 1352 (-SO2-). 1H NMR (200 MHz, d6-DMSO, d): 3.09
(br s, 4H, Pip-4H), 3.66 (br s, 4H, Pip-4H), 7.02–7.10 (m, 1H, BZT-H6), 7.21–7.29 (m, 1H,
BZT-H5), 7.43 (d, J = 8.2Hz, 1H, BZT-H4), 7.74 (d, J = 7.4 Hz, 1H, BZT-H7), 7.87 (ABd,
J = 8.0 Hz, 2H, ArH2,6), 8.5 (ABd, J = 8.4Hz, 2H, ArH3,5).
General synthesis of the hydroxamic acid derivatives 8a-d and 9a-d [19]
For the synthesis of compounds 8a-d and 9a-d a solutions of acids 6a-d and 7a-d were pre-
pared to which is added a solution of hydroxylamine.
Preparation of acid anhydride. To a solution carboxylic acid derivatives 6a-d and 7a-d (1
equivalent) in 30 mL dry THF at 0°C is added ethylchloroformate (1.2 equivalents), N-methyl-
morpholine (1.3 equivalents) with the mixture stirred for a further 1 h and filtered. The reac-
tion was controlled with TLC (PE/EtAc 3:7).
Preparation of free hydroxylamine. To a solution of potassium hydroxide (3 equivalents)
in 25 mL dry EtOH was cooled to 0°C and hydroxylamine HCl (3 equivalents) was added in
small portions After the addition the mixture was stirred for a further 1 h at 0°C and the pre-
cipitated KCl was filtered.
Preparation of target hydroxamic acid derivatives
The prepared acid anhydride in A was added dropwise to the freshly prepared hydroxylamine
solution in EtOH at 0°C. The reaction was stirred further for 1 h and then at room temperature
till the reaction declared complete after TLC control (PE/EtAc 3:7). After complete reaction
(1 h.), the mixture was evaporated to dryness and the residue taken up in cooled distilled water
and stirred for 30 min., the precipitate filtered under suction and washed with petrolether. The
products were dried in high vacuum at 60°C.
3-[4(1H-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-N-hydroxy-benzamid (8a): Benzoic acid
derivative 6a (0.270 g, 6.98 mM), N-methylmorpholine (0.092 g, 9.07 mM), ethylchloroformate
(0.091 g, 8.38 mM), KOH (0.118 g, 2,09 mM) and hydroxylamine HCl (0.146 g, 2,094 mM)
was reacted to give colourless crystals (0.205 g, 73%) of compound 8a (m. pt. 349–350°C). IR
(KBr, v cm-1): 3423 (-OH), 2982–3068 (-NH), 1613 (C = O), 1344 (-SO2) 1H NMR (200 MHz,
d6-DMSO, d): 3.08 (br. s., 4H, 4x Pip-4H), 3,62 (br s, 4H, 4x Pip-4H), 6,95 (br s, 2H, BZI-H5,6),
7.19 (br s, 2H, BZI-H4,7), 7.74–7.82 (m, 1H, ArH), 8.00–8.04 (m, 1H, ArH), 8.21–8.24 (m, 2H,
2xArH), 10.40 (br s, 1H, OH), 11.2 (br s, 1H, NH). Anal. Calcd. for C18H19N5O4S (401.44): C,
53.85; H, 4.77; N, 17.45. Found: C, 54.20; H, 5.01; N, 17.32.
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Novel HDAC6 Inhibitors
3-[4-(1-Methyl-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-N-hydroxy-benzamid (8b): Benzoic
acid derivative 6b (0.450 g, 1.12 mM), N-methylmorpholine (0.147 g, 1.46 mM), ethylchloro-
formate (0.146 g, 1.34 mM), KOH (0.188 g, 3.36 mM) and hydroxylamine HCl (0.233 g,
3.36 mM) was reacted to give colourless crystals (0.405 g, 87%) of compound 8b (m. pt. 167–
169°C). IR (KBr, v cm-1): 3432–3349 (-OH), 2854–3068 (-NH), 1615 (C = O), 1597 (C = C,
C = N), 1349 (-SO2-). 1H NMR (200 MHz, d6-DMSO, d): 3.13 (br s, 4H, 4x Pip-4H), 3.34
(br s, 4H, 4x Pip-4H), 3.51 (s, 3H, N-CH3), 7.05–7.09 (m, 2H, BZM-H5,6), 7.29–7.40 (m, 2H,
BZM-H4,7), 7.73–7.95 (m, 1H, ArH), 8.02–8.12 (m, 1H, ArH), 8.23–8.29 (m, 2H, 2x ArH),
9.25 (s, 1H, OH), 11.55 (s, 1H, NH). Anal. Calcd. for C19H21N5O4S (415.47): C, 54.93; H, 5.09;
N, 16.86. Found: C, 55.20; H, 5.30; N, 16.90.
3-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (8c): Benzoic acid deriva-
tive 6c (0.450 g, 1,16 mM), N-methylmorpholine (0.153 g, 1,51 mM), ethylchloroformate
(0.151 g, 1.39 mM), KOH (0.195 g, 3.48 mM)) and hydroxylamine HCl (0.242 g, 3.48 mM) was
reacted to give colourless crystals (0.402 g, 86%) of compound 8c (m. pt. 257–262°C). IR (KBr,
v cm-1): 1580 (C = C, C = N), 1348 (-SO2-). 1H NMR (200 MHz, d6-DMSO, d): 3.09 (br s, 4H,
4x Pip-H), 3.70 (br s, 4H, 4x Pip-H), 6.96–7.03 (m, 1H, BZO-H5 or 6), 7.08–7.16 (m, 1H,
BZO-H5 or 6), 7.24–7.27 (m, 1H, BZO-H7), 7.34–7.38 (m, 1H, BZO-H4), 7.69–7.81 (m, 1H,
ArH), 7.87–8.08 (m, 2H, 2x ArH), 8.20–8.25 (m, 1H, ArH), 9.23 (br s, 1H, OH), 11.50 (br s,
1H, NH). Anal. Calcd. for C18H18N4O5S (402.42): C, 53.72; H, 4.51; N, 13.92. Found: C, 53.79;
H, 4.57; N, 13.89.
3-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (8d): Benzoic acid deriv-
ative 6d (0.400 g, 0,98 mM), N-methylmorpholine (0.130 g, 1,27 mM), ethylchloroformate
(0.128 g, 1.18 mM), KOH (0.166 g, 2.94 mM) and hydroxylamine HCl (0.206 g, 3.36 mM) was
reacted to give colourless crystals (0.332 g, 80%) of compound 8d (m. pt. 207–210°C). IR (KBr,
v cm-1): 2949–3298 (-NH, -OH), 1653 (C = O), 1591, 1532 (C = C, C = N), 1350 (-SO2-). 1H
NMR (200 MHz, d6-DMSO, d): 3.08 (br s, 4H, 4x Pip-H), 3:67 (br s, 4H, 4x Pip-H), 7.01–7.09
(m, 1H, BZT-H6), 7.21–7.29 (m, 1H, BZT-H5), 7.72–7.76 (m, 2H, BZT-H7, Ar-H), 7.41–7.45
(m, 1H, BZT-H4), 7.87–7.91 (m, 1H, Ar-H6), 8.04–8.08 (m, 2H, Ar-H2,4), 8.85–9.65 (br s, 1H,
OH), 11.12–11.62 (br s, 1H, NH). Anal. Calcd. for C18H18N4O4S2 (418.49): C, 51.66; H, 4.34;
N, 13.39. Found: C, 51.72; H, 4.32; N, 13.26.
4-[4(1H-Benzimidazol-2-yl)-pipreazin-1-sulfonyl]-N-hydroxy-benzamid (9a): Benzimidazol
derivate 7a (0.400 g, 1.03 mM), N-methylmorpholine (0.136g, 1.34 mM), ethylchloroformate
(0.134 g, 1.24 mM), KOH (0.174 g, 3.09 mM) and hydroxylamine HCl (0.215 g, 3.09 mM) was
reacted to give colourless crystals (0.340 g, 82%) of compound 9a (m. pt. 382–383°C, decompo-
sition). IR (KBr, v cm-1): 2859-3077(-NH, -OH), 1644 (C = O), 1611 (C = N), 1360 (-SO2-).
ESI-MS (m/z): 402.14 [M+1]. 1H NMR (200 MHz, d6-DMSO, d): 3.15 (br. s, 4H, 4x Pip-4H),
3.35 (br. s, 4H, 4x Pip-4H), 6.90–6.94(m, 2H, BZI-H5,6), 7.17–7.22 (m, 2H, BZI-H4,7), 7.90
(ABd, J = 8.4Hz, 2H, ArH-2/6), 8.15 (ABd, J = 8.4Hz, 2H, ArH-3/5), 9.23 (br.s, 1H, OH), 11.50
(br.s, 1H, NH). Anal. Calcd. for C18H19N5O4S (401.44): C, 53.85; H, 4.77; N, 17.45. Found: C,
54.07; H, 4.91; N, 17.48.
4-[4-(1-Methyl-Benzimidazol-2-yl)-piperazin-1-sulfonyl]-N-hydroxy-benzamid (9b): Benzoic
acid derivative 7b (0.450 g, 1,12 mM), N-methylmorpholine (0.147 g, 1,46 mM), ethylchloro-
formate (0.146 g, 1,34 mM), KOH (0.189 g, 3,37 mM) and hydroxylamine HCl (0.234 g,
3,37 mM) was reacted to give colourless crystals (0.417 g, 89%) of compound 9b (m. pt. 269–
271°C). IR (KBr, v cm-1): 3379 (-OH), 2855–3188 (-NH), 1615 (C = O), 1353 (-SO2). 1H NMR
(200 MHz, d6-DMSO, d): 3.14 (br s, 4H, 4x Pip-4H), 3.33 (br s, 4H, 4x Pip-4H), 3.52 (s, 3H,
N-CH3), 6.98–7.14 (m, 2H, BZM-H5,6), 7.25–7.45 (m, 2H, BZM-H4,7), 7.84–7.93 (m, 2H,
2xArH), 7.98–8.02 (m, 1H, ArH), 8.17–8.21 (m, 1H, ArH), 9.29 (br s, 1H, OH), 11.50 (br s, 1H,
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Novel HDAC6 Inhibitors
NH). Anal. Calcd. for C19H21N5O4S (415.47): C, 54.93; H, 5.09; N, 16.86. Found: C, 55.18; H,
5.27; N, 16.95.
4-(4-Benzoxazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (9c): Benzoic acid deriva-
tive 7c (0.264 g, 0,681 mM), N-methylmorpholine (0.102 g, 0,89 mM), ethylchloroformate
(0. 101 g, 0,82 mM), KOH (0.130 g, 2,04 mM) and hydroxylamine HCl (0.161 g, 2,04 mM)
was reacted to give colourless crystals (0.225 g, 82%) of compound 9c (m. pt. 335–338°C). IR
(KBr, v cm-1): 3400 (-OH), 2869–3094 (-NH), 1689 (C = O), 1582 (C = N), 1346 (-SO2-). 1H
NMR (200 MHz, d6-DMSO, d): 3,10 (br s, 4H, 4x Pip-4H), 3.70 (br s, 4H, 4x Pip-4H), 7.00
(t, J = 7.2Hz, 1H, BZO-H5 or 6), 7.13 (t, J = 7.2Hz, 1H, BZO-H5 or 6), 7.26 (d, J = 7.2Hz, 1H,
BZO-H7), 7.36 (d, J = 7.4Hz, 1H, BZO-H4), 7.81–7.89 (m, 2H, 2x ArH), 7.97 (d, J = 8.2Hz, 1H,
ArH), 8.15 (d, J = 7.6Hz, 1H, ArH), 9.25 (br s, 1H, OH), 11.45 (br s, 1H, NH). Anal. Calcd. for
C18H18N4O5S (402.42): C, 53.72; H, 4.51; N, 13.92. Found: C, 53.83; H, 4.55; N, 13.91.
4-(4-Benzothiazol-2-yl-piperazin-1-sulfonyl)-N-hydroxy-benzamid (9d): Benzoic acid deriv-
ative 7d (400 mg, 0,99 mM), N-methylmorpholine (130,36mg, 1,29 mM), ethyl chloroformate
(129,11 mg, 1,19 mM), KOH (166,87 mg, 2,97 mM) and hydroxylamine HCl (206.67 mg,
2.97 mM) was reacted to give colourless crystals (0.343 g, 82%) of compound 9d (m. pt. 324°C,
decomposition). IR (KBr, v cm-1): 2858–3046 (-NH, -OH), 1689/1639 (C = O), 1593 (C = N),
1543 (C = C),1350 (-SO2-) 1H NMR (200 MHz, d6-DMSO, d): 3.09 (br s, 4H, 4x Pip-4H),
3.66 (br s, 4H, Pip-4H), 7.06 (t, J = 7.2Hz, 1H, BZT-H6), 7.26 (t, J = 7.4Hz, 1H, BZT-H5), 7.43
(d, J = 7.4Hz, 1H, BZT-H4), 7.74 (d, J = 7.4Hz, 1H, BZT-H7), 7.83–7.87 (m, 2H, 2x ArH), 7.97
(d, J = 8.6Hz, 1H, ArH), 8.15 (d, J = 7.8Hz, 1H, ArH), 10.40 (br s, 1H, OH), 11.20 (br s, 1H,
NH). Anal. Calcd. for C18H18N4O4S2 (418.49): C, 51.66; H, 4.34; N, 13.39. Found: C, 51.79; H,
4.39; N, 13.40.
LDR studies and measures of GFP. LDR cells were maintained in DMEM with L-gluta-
mine and high glucose, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 1%
v/v penicillin-streptomycin and 10% heat inactivated FBS. Cells were seeded on glass bottom
chambers at low-medium density and allowed to attach overnight. Compounds were then
added for 24h at the indicated dose and cells were imaged under a fluorescent microscope
equipped with a camera using a 20 or 40X objective. All images shown were exposed for identi-
cal time.
Purification of HDAC1 and Initial Histone Deacetylase Assay. For inhibition assays,
partially purified HDAC1 (70–80 fold enriched relative to crude samples) from mouse A20
cells was used as enzyme source. HDAC1 activity was purified by a combination of anion
exchange chromatography (Mono Q), affinity chromatography (Poly-lysin agarose, Heparin
Sepharose) and TSK size exclusion chromatography (Jesacher and Loidl, unpublished).
HDAC1 protein was identified by immunoblotting using anti mouse HDAC1 antibodies (col-
laboration with Zymed Laboratories, Inc., San Francisco, California, USA). HDAC activity was
determined as described by Sendra et al., [20] using [3H]acetate-prelabeled chicken reticulocyte
histones as substrate. 50 μL of mouse HDAC1 were incubated with different concentrations of
compounds for 10 min on ice and 10 μL of total [3H] acetate pre-labeled chicken reticulocyte
histones (4 mg/mL) were added, resulting in a concentration of 41 μM. This mixture was incu-
bated at 37°C for 1 h. The reaction was stopped by addition of 50 μL of 1 M HCl/0.4 M acetyla-
cetate and 1 mL ethylacetate. After centrifugation at 10000 x g for 5 min an aliquot of 600 μL of
the upper phase was counted for radioactivity in 3 mL liquid scintillation cocktail.
MTS viability assays. Lung cancer cells (kindly provided by Dr. John D. Minna, [13, 21])
were maintained in RPMI 1640 media supplemented with 10% FBS. Immortalized normal
human bronchial epithelial cells (also provided by Dr. John D. Minna [13, 21]) were cultured
in KSFM supplemented with pituitary extract and EGF. For viability assays, cells were plated
on 96 well plates and grown overnight. Cells were then exposed to increasing doses of the
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Novel HDAC6 Inhibitors
indicated compounds for 4 days and their viability assayed using the Promega Cell Titer 96
AQueous One kit per the manufacturer’s protocol. All doses were tested in 4–8 replicates. IC50
values were calculated using the in-house software DIVISA.
Cell cycle and apoptosis assays. For cell cycle analysis, cells were washed with PBS, fixed
in 70% ethanol for 2 h, washed with PBS and resuspended in staining solution (0.1% Triton X-
100, 0.2 mg/ml DNAse-free RNAse A and 20 mg/ml PI) and incubated at 37C for 15 min. His-
tograms were acquired on a BD FACSCalibur flow cytometer at UT Southwestern’s core facil-
ity. Apoptosis was measured using a FITC Annexin V Apoptosis Detection Kit I (BD
Pharmingen) according to manufacturer’s protocol after treating cells with compounds as indi-
cated in Figure legends.
Comparative HDAC inhibition assays. For the direct comparison of HDAC1 vs. HDAC6
inhibition, HDAC activity was analyzed using the HDAC Assay Kit from Millipore (17–356).
Briefly, recombinant HDAC1 or recombinant HDAC6 (both purchased from Epigenetek,
Purity of HDAC1 is more than 60% and purity HDAC6 is more than 85%). was incubated with
the fluorometric HDAC substrate according to the manufacturer’s protocol in the presence of
DMSO vehicle or increase compound doses. In a secondary activator reaction, the fluorophore
is only cleaved from the deacetylated substrate, allowing for quantification. Fluorescence was
quantified on a FLUOstar-Optima or a FLUOstar Omega plate reader (BMG Biosciences). To
ensure the compounds do not interfere with the detection system control experiments with
standard deacetylated substrate were performed in the presence of high doses of all
compounds.
Computational methods. The crystal structure of human HDAC1 [22] (PDB ID: 4BKX)
and a homology model of HDAC6 that is described elsewhere [23] was used for the docking
study. The protein structure for docking was prepared using the Protein Preparation Wizard
[24] within the Schrödinger software package Schrödinger Inc, New York, USA. The hydrogen
bond network was optimized automatically and the protonation states were predicted with the
PROPKA tool at pH 7.0. Ligands were prepared in MOE [25] (version 2012.10, Chemical
Computing Group, Montreal, Canada). Conformational search for all structures has been car-
ried out using the Low Mode MD sampling with a minimum RMSD between the conforma-
tions of 1 Å. This procedure was applied to obtain realistic conformations for all ligands
containing flexible ring systems. Molecular docking studies have been performed on a Linux
cluster using the Glide software (Schrödinger Inc, New York, USA) [24] in standard precision
(SP) mode. Constraints to the Zn2+ ion were set and two conserved water molecules located
inside the binding pocket were considered during the docking. Post-docking filtering has been
carried out using the Python script distance_to_smarts (Revision 3.5, Schrödinger). Only dock-
ing poses with distances between both oxygen atoms of the hydroxamic acid of the ligand and
the Zn2+ ion inside the binding pocket of the protein below 2.7 Å have been selected. Rescoring
of the docking poses was done applying the MM-GBSA approach. The MMF94 force field and
a GBSA solvation model within the MOE software package were used. Ligands were minimized
inside the binding pocket until a gradient of 0.05 kcal/mol was reached. Protein heavy atoms
were tethered using a force constant of 100 kcal/mol Å.
Results
Identification of 9b as an LDR active with HDAC inhibitory activity
We incubated LDR cells with compounds in our collection at 20μM and measured the activa-
tion of the silenced GFP gene. Fluorescence microscopy showed robust GFP expression in
response to 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydro
xamic acid (9b) treatment (Fig 2A). We confirmed that the signal was not coming from
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Novel HDAC6 Inhibitors
Fig 2. Structure and locus de-repression assay activity of Trichostatin A (TSA) and 9b-9d. A, Structure
of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydroxa-mic acid, (compound 9b). B,
LDR cells were assayed for GFP expression by fluorescence microscopy after overnight treatment with the
indicated doses of the DMSO vehicle, the trichostatin A positive control or 9b-9d, followed by estradiol
induction of the nuclear translocation of the GFP-estrogen receptor chimeric transgene. DAPI staining of the
nucleus is shown for reference. The other compounds were inactive in the LDR assay.
doi:10.1371/journal.pone.0134556.g002
compound auto-fluorescence by co-inducing with estradiol which causes the nuclear transloca-
tion of the GFP chimeric protein [26]. Thus, 9b is a bona fide activator of the expression
of GFP in the LDR system, suggesting that it may have activity as an epigenetic modulator
(Fig 2B).
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Novel HDAC6 Inhibitors
Fig 3. Structure of R306465, an HDAC inhibitor developed by Johnson & Johnson that shares
structural motifs with 9a-9d series.
doi:10.1371/journal.pone.0134556.g003
Since the LDR assay is sensitive to hydroxamic acid HDAC inhibitors we hypothesized that
9b may be acting by this mechanism. Furthermore, compound 9b has the structural elements
that have been described as a common template for HDAC inhibitors, comprising a cap group
(1-methylbenzimidazole), a zinc binding enzyme inhibition group (CO(NHOH) functionality)
and a spacer (piperazine-sulfonylbenzene moiety) that links the cap to the binding system [27].
Moreover, an HDAC inhibitor, R306465 (Fig 3) with juxtaposed flanking groups developed by
Johnson & Johnson [28] shares significant sub-structural similarity to 9b and its analogs.
To evaluate this directly, we performed in vitro HDAC1 activity assays using a partially
purified system with A20 cells as the source of enzyme. As can be seen in Fig 4, 9b is indeed an
HDAC inhibitor with an IC50 of approximately 12μM in this assay, and its structure may be
used as a scaffold to further diversify this activity. We therefore explored the structure/activity
relationship of two elements of 9b by synthesizing and analyzing the effects of the replacement
of the 1-methylbenzimidazole ring by the isosteric heterocycles benzimidazole, benzoxazole
and benzothiazole and a variation of the position of the hydroxamic acid substituent on the
phenyl ring (3- and 4- position (see Fig 1)).
Locus derepression activity and ability of 9b analogues to inhibit HDAC1
activity
Using the LDR cells, we first assayed the 9b analogues 8a-d, 9a, 9c and 9d for their ability to
induce the GFP transgene in our system. Indeed, a high level of GFP induction was observed
Fig 4. Induction of GPF and inhibitory activity against partially purified HDAC1. NS = not soluble,—
negative, *The mean values of at least two independent experiments in which duplicate determinations were
taken.
doi:10.1371/journal.pone.0134556.g004
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Table 1. Antiproliferative activity (IC50 μM) of compounds 9a-d, TSA, Apicidin and Depsipeptide.
Compd
HCC4017
HBEC-30KT
Selectivity
HCC4018
HBEC-34KT
Selectivity
9a
9b
> 10*
1.49
> 10
> 10
-
>10
6.1
>10
> 10
-
> 6.71
> 8.06
3.29
3.90
0.75
0.14
> 1.64
> 1.98
2.06
9c
1.24
> 10
5.05
4
> 10
9d
1.63
5.37
8.22
TSA
Apicidin
Depsi.
0.073
0.12
0.285
0.09
0.023
0.03
0.0042
0.346
0.79
15.0
26.3
0.0065
0.0009
0.0013
0.31
Patient A
Patient B
*The mean values of at least three independent experiments in which duplicate determinations were taken within each experiment. For clarity, standard
deviation has been omitted.
doi:10.1371/journal.pone.0134556.t001
upon treatment of the cells with compounds 9c, 9d (Fig 2B) which bears a hydroxamic acid
functionality at the four position. In this assay system, compounds 8a-d bearing the hydroxa-
mic acid functionality in the three position of the phenyl ring did not induce GFP (Fig 4). The
position of the hydroxamic acid thus appears critical to activity and is likely related to the abil-
ity of this group to reach into the catalytic site in order to chelate the zinc ion. In addition, com-
pound 9a where X = NH was also inactive in the LDR assay likely due to poor cell
permeability.
We then evaluated if the 9b analogues which were LDR actives were able to inhibit HDAC1
activity. For enzyme inhibition assays, partially purified HDAC1 from mouse A20 cells was
used as enzyme source. Compared to 9b (IC50 = 12.4 μM), both 9c (IC50 = 5.7 μM) and 9d
(IC50 = 4.8 μM) were two to three fold more potent in inhibiting HDAC1 activity (Table 1).
Although compound 9a was inactive in the LDR assay, it is equipotent (IC50 = 12.4 μM) com-
pared to 9b in the in vitro HDAC inhibition assay, again suggesting poor cell penetration in the
LDR assay. As a control, we also measured LDR inactives 8a-d for their HDAC1 inhibitory
potential and found that as expected they were 10–60 fold less potent in vitro than 9b (Fig 4).
Anti-proliferative activity and cancer-selectivity of compounds 9a- 9d
Given the known anti-cancer qualities of HDAC inhibitors, we evaluated the most potent com-
pounds for their ability to block cancer cell proliferation and for their selectivity for cancer vs.
normal cells. To accomplish this, we performed standard MTS viability assays on two pairs of
patient matched non-small cell lung cancer cells and their corresponding normal human epi-
thelial lines. As can be seen in Table 1, compounds 9b, 9c and 9d all had anti-proliferative
activity with IC50 values between 1–6 μM. Furthermore, these three compounds demonstrated
cancer selectivity, affecting the cancer lines HCC4017 and HCC4018 several fold more potently
than the patient matched HBEC normal lines HBEC30KT and HBEC34KT, respectively
(Table 1). Again, we saw no cellular activity with 9a.
Compared to the known HDAC inhibitors TSA, apicidin and depsipeptide, compounds 9b,
c and d had less potent antiproliferative activity (Table 1). On the other hand, compounds 9b-
d showed better selectivity for patient A matched cells compared to the three known HDAC
inhibitors. However, for cells derived from patient B TSA and apicidin exhibited significantly
higher selectivity by a factor of 7–13 fold compared to compounds 9b-d, yet in both paired cell
lines depsipeptide was more toxic to normal cells than to the patient-matched cancer cells.
PLOS ONE | DOI:10.1371/journal.pone.0134556 December 23, 2015
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Novel HDAC6 Inhibitors
Effect of compounds 9b and 9c on cell cycle progression and induction
of apoptosis
Recently, HDAC inhibitors have emerged as promising chemotherapeutic agents, and the find-
ings of several studies suggest that they can induce a range of anti-tumor activities including
the induction of cell-cycle arrest, the stimulation of differentiation, and the induction of apo-
ptosis in a variety of transformed cells in culture. [29] In agreement with this, we found that
compounds 9b and 9c affect cell cycle progression. FACs analysis indicated that both 9b and
9c affect cell cycle distribution after just 18h of treatment. Indeed the higher doses markedly
influenced S-phase progression and resulted in accumulation of cells in S/G2/M especially at
later times (Fig 5A and 5B). Compound 9c had particularly robust effects on cell cycle phase
distribution (Fig 5B). Of note, no subG0 population was observed in either 9b or 9c treated
cells even after 64h. Consistent with this, Annexin V staining did not show any reproducible
significant changes in the percent of dead cells (S1–S3 Figs).
Tissue-specific antiproliferative activity in tumor cell lines
To evaluate if the antiproliferative activity of the novel hydroxamic acid derived compounds
was general across cancer types, selected compounds were submitted for testing in the tissue-
specific panel of human tumor cell lines at the National Cancer Institute, Bethesda, USA. This
screen utilizes 60 human tumor cell lines, representing leukemia, melanoma and cancers of the
lung, colon, brain, ovary, kidney, prostate and breast [30]. This cell line panel offers the advan-
tage of having available microarray gene expression data across the 60 cell lines which can be
used to determine the uniqueness of a new lead for which the molecular target(s) might not be
known. The antitumor activity is presented for each cell line by three parameters, namely log10
Fig 5. A, B: 9b and 9c strongly alter cell cycle distribution of cancer cells. HCC4017 lung cancer cells show defects in S-phase progression and
accumulation in G2/M after treatment with 9b (in A) or 9c (in B), as indicated. Cell cycle histograms were collected on PI stained samples of floating and
adherent cells, on a FACs Calibur.
doi:10.1371/journal.pone.0134556.g005
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Novel HDAC6 Inhibitors
GI50 value (GI50 = molar concentration of the compound that inhibits 50% net cell growth),
log10 TG value (TGI = molar concentration of the compound leading to total inhibition of net
cell growth), and log10 LC50 value (LC50 = molar concentration of the compound leading to
50% net cell death) accompanied by MID which refers to the average inhibitory value across
the 60 cell lines.
Compounds 9b-d which were active in the in vitro HDAC assay and showed inhibition of
cancer vs. normal lung cells, were submitted for testing across the 60 cell line panel for antipro-
liferative activity at a single dose of 10 μm in the initial screening assay. Only compounds 9c
and 9d exhibited significant growth inhibition at this dose (average 50% and 62% respectively).
These results are summarized in S4–S6 Figs. The activity of these two agents was then tested
over a five dose concentration curve. In this assay, compounds 9c (NSC 747073) and 9d (NSC
747072) inhibited the proliferation of subsets of leukemia, colon, CNS, melanoma, and pros-
trate cancer cells to a higher extent than cells from other cancers (Fig 6A and 6B). As shown in
Table 2, of the two compounds, 9d was more potent overall by a factor of 2 (MID: = 3.16 μm),
compared to 9c (MID: = 6.61 μM) consistent with its higher activity in the one dose assay.
Fig 6. Differential response of cancers to 9c and 9d. A, GI50 values across the NCI-60 cell line panel in response to 9c. B, GI50 values across the NCI-60
cell line panel in response to 9d.
doi:10.1371/journal.pone.0134556.g006
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Novel HDAC6 Inhibitors
Modeling of 8a-d and 9a-d in the catalytic site of HDACs
Molecular docking was carried out using the X-ray structure of human HDAC1 [22] and a
homology model of human HDAC6 [23] to investigate the interaction of compounds 8a-d and
9a-d with these enzymes and explore potential specificity differences since these two HDACs
have distinct biology and their catalytic sites show differences that can be targeted. The
obtained docking poses were re-scored using the MM-GBSA approach. For both HDAC sub-
types, the para-substituted inhibitors 9a-d showed favorable interactions (Fig 7 and Fig 8). The
hydroxamic acid group is chelating the zinc ion and makes a hydrogen bond to His141 in
HDAC1 and His611 in HDAC6. The aromatic ring of the inhibitors interacts with two con-
served Phe residues (Phe150 and Phe 205 in HDAC1, Phe620 and Phe680 in HDAC6) whereas
the sulfonamide group interacts with a conserved water molecule observed in HDAC crystal
structures. Only in case of the para-substituted derivatives, the terminal bicyclic aromatic ring
system makes favorable interaction with the rim of the catalytic pocket (Gly27, His28 and
Pro29 in HDAC1, His500, Pro501 and Tyr570 in HDAC6). In case of HDAC6, a perfect fit
between the benzimidazole, benzoxazole and benzothiazole group of 9a-d with the surface of
the catalytic pocket is observed (Fig 8) which would predict stronger inhibition of HDAC6
over HDAC1. For the meta-substituted derivatives (8a-d), which are inactive or weakly active,
the terminal group is extending in to the solvent (Fig 9).
In case of HDAC1, the MM-GBSA energies were able to discriminate between actives (9a-
d) and weakly active compounds (8a-d) (Table 3). For the most potent HDAC6 inhibitors
favorable MM-GBSA energies were derived (Table 3).
The para-substituted compounds 9a-d show HDAC6 selectivity
To directly test if 9b and its analogs had greater potency to inhibit HDAC6 (a class IIb enzyme)
compared to HDAC1 a (class I enzyme) as predicted in the virtual ligand binding assay, we
performed parallel experiments for both enzymes using purified proteins and measured activ-
ity using a fluorophore system designed by Millipore (see Methods) which quantifies the
amount of acetylated histone substrate. Although we found no differences in IC50 for HDAC6
vs. HDAC1 in the meta-substituted compounds (data not shown), a large difference in IC50
was apparent for the para-substituted compounds (Fig 10). In all cases, the potency against
Table 2. Inhibitory concentration values across histological cancer types.
NSC 747073 (9c)
NSC 747072 (9d)
Cancer Type
GI50 (μM)
GI50 (μM)
Leukemia
2.24 (4/4)b
9.51 (3/9)
6.05 (4/6)
5.82 (4/6)
5.62 (6/8)
7.66 (2/5)
8.24 (3/8)
4.89 (2/2)
8.71 (2/7)
6.61
1.77 (5/5)
3.77 (4/9)
2.30 (6/7)
2.77 (4/6)
2.66 (5/8)
4.01 (3/3)
3.94 (3/8)
2.48 (2/2)
3.88 (4/7)
3.16
NSCL cancera
Colon cancer
CNS cancer
Melanoma
Ovarian cancer
Renal Cancer
Prostate Cancer
Breast cancer
MIDc
aNon-Small Cell Lung cancer
bFigures in parenthesis refers to no. cell lines used for the assay/no inhibited by compound
cAverage value of inhibition across the 60 cell lines
doi:10.1371/journal.pone.0134556.t002
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Novel HDAC6 Inhibitors
Fig 7. Docking poses of compounds 9a-d in the HDAC1 catalytic domain. Inhibitors are colored orange.The zinc ion is shown as a brown ball. Hydrogen
bonds between HDAC1 and inhibitors are shown as blue lines and distances are given in Å. On the right side, the molecular surface is displayed and
contoured according to the hydrophobic potential (magenta = hydrophilic, green = hydrophobic).
doi:10.1371/journal.pone.0134556.g007
HDAC6 was 5–30 fold greater than that against HDAC1 (Table 4). However, compounds 9a-d
are less potent as HDAC 6 inhibitor compared to Tubastatin A [31] and TSA [32].
Thus, we have characterized novel compounds 9a-d as HDAC6 selective inhibitors in vitro
and shown that three of them (9b, 9c and 9d) have specific antiproliferative activity against
cancer but not normal cells.
Fig 8. Docking poses of compounds 9a-d in the HDAC6 catalytic domain. Inhibitors are colored in cyan. The zinc ion is shown as a brown ball. Hydrogen
bonds between HDAC6 and inhibitors are shown as blue lines and distances are given in Å. On the right side the molecular surface is displayed and
contoured according to the hydrophobic potential (magenta = hydrophilic, green = hydrophobic).
doi:10.1371/journal.pone.0134556.g008
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Novel HDAC6 Inhibitors
Fig 9. Docking results for compounds 8a-8d in HDAC1. Due to the meta-substitution of the sulfonamide
linker the terminal group of the inhibitors is not able to favorably interact with the rim of the pocket. The
molecular surface is displayed and contoured according to the hydrophobic potential (magenta = hydrophilic,
green = hydrophobic).
doi:10.1371/journal.pone.0134556.g009
Discussion
Using an established cell based assay to screen for epigenetic modulators, we report the identi-
fication of a novel series of hydroxamic acid derivatives which possess activity as HDAC inhibi-
tors selectively inhibiting HDAC6 over HDAC1 and having specific antiproliferative activity
on human cancer cells vs. patient matched normal cells. Compounds with the hydroxamic acid
functionality in the para-position of a benzene ring exhibited favorable HDAC inhibitory and
antiproliferative activities. Although the potency of these compounds was lower compared to
known HDACi TSA, apicidin and depsipeptide, they had comparable or greater selectivity of
inhibition for cancer vs. normal cells and for HDAC6 vs HDAC1. In contrast, the meta substi-
tuted analogues were devoid of biological activity. The antiproliferative effect of 9b and 9c was
at least in part mediated by robust changes in the cell cycle distribution leading to growth arrest
of cancer cells exposed to these compounds, in line with known effects of HDAC inhibition.
Table 3. Inhibitory activities and MM-GBSA energies of the studied compounds.
Compound
HDAC1 IC50 [μM]
HDAC1 pIC50
MM-GBSA (kcal/mol)
HDAC6 IC50 [μM]
HDAC6 pIC50
MM-GBSA(kcal/mol)
8a
8b
8c
8d
9a
9b
9c
9d
n.i.
-
-20.44
-20.77
-20.89
-20.96
-29.51
-28.27
-28.88
-29.43
-
-
n.c
316
216
140
12.4
12.4
5.7
3.50
3.67
3.85
4.91
4.91
5.24
5.32
-
-
n.c.
-
-
n.c.
-
-
n.c
1.00
0.10
0.20
0.10
6.00
7.00
6.70
7.00
-34.72
-36.03
-35.39
-36.47
4.8
n.c. = not calculated, n.i. = no inhibition
doi:10.1371/journal.pone.0134556.t003
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Novel HDAC6 Inhibitors
Fig 10. Compounds 9a-9d inhibit HDAC6 over HDAC1. Dose response curves of 9a-9d comparative inhibition of HDAC6 vs. HDAC1 activity using purified
enzymes for in vitro assays measuring deacetylation of histone substrates. Assays were performed in parallel for both enzymes.
doi:10.1371/journal.pone.0134556.g010
Molecular modeling studies uncovered that the MM-GBSA energy data for interaction with
HDAC6 was higher than for HDAC1 indicating that 4-[4-(1-methylbenzimidazol-2-yl)pipera-
zin-1-yl]sulfonylbenzenecarbohydroxamic acid and its analogues might be selective HDAC6
inhibitors. Confirmatory studies showed that these compounds are indeed selective HDAC6
inhibitors compared to HDAC1 by a factor 6–40. In addition, 9b-9d demonstrated potent
blocking of tumor cell viability while not affecting normal cells derived from the same patients.
Several HDACi are in preclinical development and clinical trials for the treatment of a wide
range of diseases, including cancer. The in vivo toxicities associated with some of these com-
pounds due to their molecular promiscuity may well prevent them from ever reaching clinical
Table 4. Enzyme Inhibition Data of Compounds 9a-d.
HDAC1*
IC50 (μM)
HDAC6*
IC50 (μM)
Selectivity Index
HDAC1/HDAC6
Compounds
9a
6.0 +/- 2.9
0.93 +/-0.75
6.5
9b
3.7 +/- 1.7
0.13 +/- 0.07
0.22 +/- 0.04
0.13 +/- 0.02
0.015+/-0.001
0.0086+/-0.0014
28.4
4.3
9c
0.92 +/- 0.16
0.90 +/- 0.45
16.4+/-2.6
9d
9.6
aTubastatin A
bTSA
1093
1.4
0.0060+/-0.0025
aTaken from ref. [31]
bTaken from ref. [32].
* Indicated are the mean values of at least three independent experiments.
doi:10.1371/journal.pone.0134556.t004
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Novel HDAC6 Inhibitors
efficacy. The current hypothesis is that the development of selective inhibitors targeting only
one member or a subclass of the HDAC family will lead to improved overall efficacy and fewer
side effects. In view of this, the compounds described herein, particularly 9b, can serve as lead
structures for the development of selective HDAC6 inhibitors with improved and more tar-
geted activity. HDAC6 selective inhibitors have been described including Tubacin, Tubastatin
A, ACY-1215 and several others [9, 33]. These inhibitors appear to primarily affect non-his-
tone targets of HDAC6 including α-tubulin and HSP90 and have beneficial effects in disease
models including cancer but also extending to neurodegeneration and immunity [31].
Among known HDAC inhibitors, R306465 developed by Johnson & Johnson [28] shares
significant sub-structural similarity to 9b and its analogs. The in vitro specificity of R306465
suggested a preference for HDAC1 vs. HDAC8 and cellular studies determined that both his-
tone and tubulin acetylation were increased by the compound though the effects on histone
acetylation were stronger and detected at lower doses compared to tubulin acetylation [28].
Consistent with this, HDAC6 downstream target Hsp70 was not induced except by high doses
of R306465 and c-raf was only degraded at similar high doses. Thus, R306465 appears to be
selective for HDAC1 compared to HDAC6 at least in the tested cells. Remarkably, our study
suggests that rearrangement of the chemical groups of R306465 into the 9b-d configuration
switches the selectivity of the compounds towards HDAC6. Clinical studies with R306465 have
been reported in abstract form and toxicities were moderate with some indication of target
involvement and efficacy in regimens below the maximal tolerated doses [34]. This, taken
together with our studies and the beneficial effects of HDAC6 inhibitors reported in other dis-
ease models [31, 33] indicates that 9b related compounds have clinical promise and should be
further developed.
Supporting Information
S1 Fig. HCC4017 cells were treated with 9b or 9c, as indicated, for 18h and then apoptosis
was measured using a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen)
according to manufacturer's protocol.
(PDF)
S2 Fig. HCC4017 cells were treated with 9b or 9c, as indicated, for 40h and then apoptosis
was measured using a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen)
according to manufacturer's protocol.
(PDF)
S3 Fig. HCC4017 cells were treated with 9b or 9c, as indicated, for 64h and then apoptosis
was measured using a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen)
according to manufacturer's protocol.
(PDF)
S4 Fig.
(PDF)
S5 Fig.
(PDF)
S6 Fig. In vitro data from the evaluation of compounds 9b-9d against the NCI 60 cancer
cell lines at a single dose of 10 uM. Compounds 9c and 9d which exhibited significant growth
inhibition were evaluated against the 60 cell panel at five concentration levels. This information
can be at https://dtp.cancer.gov/.
(PDF)
PLOS ONE | DOI:10.1371/journal.pone.0134556 December 23, 2015
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Novel HDAC6 Inhibitors
Acknowledgments
We thank Jianjun Chang for technical assistance with proliferation assays. This project was
partly funded by the NIH (R01CA12526901 to E.D.M., 1R21AI11622201 to I.D./E.D.M. P50-
CA70907 to J.D.M., subaward to E.D.M.) by the Doctor’s Cancer Foundation (Nolan Miller
Lung Cancer grant to E.D.M.), by the Friends of the Cancer Center, by the Welch Foundation
(grants to E.D.M.) and by CPRIT (RP120717).
Author Contributions
Conceived and designed the experiments: JE GB WS EDM. Performed the experiments: LW
MK GB JM. Analyzed the data: JE WS EDM. Contributed reagents/materials/analysis tools: JE
GB WS EDM. Wrote the paper: JE GB WS EDM.
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