Structural Requirements of Histone Deacetylase Inhibitors: SAHA Analogs Modified on the Hydroxamic Acid

Article abstract of DOI:10.1002/ardp.201500472

Histone deacetylase (HDAC) proteins have emerged as targets for anti-cancer therapeutics, with several inhibitors used in the clinic, including suberoylanilide hydroxamic acid (SAHA, vorinostat). Because SAHA and many other inhibitors target all or most o

Full text of DOI:10.1002/ardp.201500472

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Full Paper
Structural Requirements of Histone Deacetylase Inhibitors:
SAHA Analogs Modified on the Hydroxamic Acid
Anton V. Bieliauskasꢀ, Sujith V. W. Weerasingheꢀꢀ, Ahmed T. Negmeldin, and Mary Kay H. Pflum
Department of Chemistry, Wayne State University, Detroit, MI, USA
Histone deacetylase (HDAC) proteins have emerged as targets for anti-cancer therapeutics, with
several inhibitors used in the clinic, including suberoylanilide hydroxamic acid (SAHA, vorinostat).
Because SAHA and many other inhibitors target all or most of the 11 human HDAC proteins, the
creation of selective inhibitors has been studied intensely. Recently, inhibitors selective for HDAC1
and HDAC2 were reported where selectivity was attributed to interactions between substituents
̊
on the metal binding moiety of the inhibitor and residues in the 14-A internal cavity of the HDAC
enzyme structure. Based on this earlier work, we synthesized and tested SAHA analogs with
substituents on the hydroxamic acid metal binding moiety. The N-substituted SAHA analogs
displayed reduced potency and solubility, but greater selectivity, compared to SAHA. Docking
̊
studies suggested that the N-substituent accesses the 14-A internal cavity to impart preferential
inhibition of HDAC1. These studies with N-substituted SAHA analogs are consistent with the
̊
strategy exploiting the 14-A internal cavity of HDAC proteins to create HDAC1/2 selective
inhibitors.
Keywords: Docking studies / HDAC inhibitors / Histone deacetylase / Isoform selectivity / Vorinostat
Received: January 5, 2016; Revised: March 15, 2016; Accepted: March 18, 2016
DOI 10.1002/ardp.201500472
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
acid, vorinostat (SAHA, Fig. 1) gaining FDA approval in 2006
for the treatment of advanced cutaneous T-cell lymphoma
Introduction
Histone deacetylase (HDAC) proteins play an important role in
gene expression by governing the acetylation state of lysine
residues located on the amino-terminal tails of nucleosomal
histone proteins [1]. Due to their fundamental role in gene
expression, HDAC proteins have been associated with basic
cellular events and disease states, including cell growth,
differentiation, and cancer formation [2]. As a result, HDAC
proteins have emerged as attractive targets for anti-cancer
therapy. Several HDAC inhibitor (HDACi) drugs are in various
stages of clinical trials [3], with suberoylanilide hydroxamic
(CTCL) [4]. Consistent with their clinical effects, inhibitors of
HDAC proteins suppress tumor cell proliferation, induce cell
differentiation, and upregulate crucial genes associated with
anti-cancer effects, such as p21 [5]. Therefore, HDACi drugs
represent
a promising next generation of anti-cancer
therapeutics.
Many HDACi drugs in and out of clinical trials, including
SAHA, inhibit all HDAC isoforms nonspecifically (so called pan-
inhibitors) [6]. The HDAC family contains 11 metal-dependent
proteins in humans, which are separated into three classes
based on their size, cellular localization, number of catalytic
Correspondence: Prof. Mary Kay H. Pflum, Department of
Chemistry, Wayne State University, 5101 Cass Avenue, Detroit,
MI 48201, USA.
E-mail: pflum@wayne.edu
Fax: þ1-313-577-8822
ꢀCurrent address: Ash Stevens, Inc., 18655 Krause Street, River-
view, MI 48193, USA.
ꢀꢀCurrent address: Department of Molecular & Integrative
Physiology, University of Michigan Medical School, 1137 E
Catherine Street, Ann Arbor, MI 48109, USA.
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Figure 1. Structures of select HDAC inhibitors with the structural regions indicated at the top.
active sites, and homology to yeast HDAC proteins. Class I
includes HDAC1–3, and 8. Class II consists of six HDAC proteins
– HDAC4–7, 9, and 10. HDAC11 is the sole member of class IV,
based on phylogenetic analysis [7]. It is a matter of debate
whether inhibiting the entire HDAC family is required for
anti-cancer activity [8]. However, elucidating the individual
functions of HDAC protein family members is impossible using
pan-inhibitors. Selective HDAC inhibitors that affect a single
HDAC isoform (isoform-selective HDACi) would be ideal
pharmacological tools to elucidate the individual functions
of each HDAC isoform. In addition, isoform-selective HDAC
inhibitors might provide a more effective chemotherapeutic
compared to pan-inhibitors [9].
cavity [26]. A significant conclusion of these studies is
that the metal binding moiety can be modified to create
selective HDAC inhibitors.
̊
To further exploit the 14-A internal cavity for selective
inhibitor design, we created SAHA analogs functionalized on
the amine of the hydroxamic acid metal binding moiety. Like
the benzamide of compound 1 [15], crystallographic and
modeling analyses indicate that the hydroxamic acid is
positioned at the base of the active site channel adjacent
to the internal cavity [11–19, 27]. Given the HDAC1/2
selectivity of compound 1 [26], we hypothesized that alkyl
or aryl groups attached to the hydroxamic acid of SAHA would
also impart selectivity.
Toward creating isoform selective inhibitors, the three
structural regions of the inhibitors (Fig. 1) have been
modified, focusing primarily on the capping region and
metal binding moiety [10]. The high sequence similarity
within the active sites of the isoforms makes inhibitor
design problematic [10]. Recently, the structures of
HDAC1–4, 7, and 8 were reported [11–17], along with
homology models of the other HDAC isoforms [18, 19].
Results and discussion
Inhibitor synthesis
To create N-modified SAHA analogs, suberic acid 3 was
refluxed in acetic acid to produce suberic anhydride 4
(Scheme 1), with subsequent ring opening with aniline to
̊
According to structural analysis, a 14-A internal cavity exists
produce 5. Separately, O-benzylhydroxylamine
6
was
deep within the HDAC active site near the catalytic metal
atom, which functions as an exit channel for release of the
acetate byproduct after acetyl-lysine deacetylation [20–22].
Important for inhibitor development, several compounds
have been designed to target the internal cavity by
appending large aromatic groups to the metal binding
moiety [15, 23, 24]. For example, compound 1 (Fig. 1)
displays selectivity for HDAC1 and HDAC2 compared to
HDAC3–8 [25, 26]. Docking studies of 1 into the HDAC1 and
HDAC3 homology models suggested that selectivity was
due to differential interactions of the aryl group on the
Boc-protected and then alkylated with various alkyl and
aryl halides to produce 8. The Boc group on N-Boc,
O-benzylhydroxylamine 7 was necessary to increase the
acidity of the amine proton and facilitate alkylation [28].
However, alkylation of hydroxyl amine 6 with a benzyl group
proceeded directly to give the O,N-dibenzyl hydroxyl amine 9,
which was subsequently coupled with suberoylanilide acid 5
to produce O-benzyl protected analogs 10c. For the other
analogs, deprotection of 8 using TFA and neutralization was
necessary prior to coupling. Finally, hydrogenolysis of 10a–e
afforded removal of the O-benzyl protecting group to give
hydroxamic acids 2a–e.
̊
metal binding group with residues in the 14-A internal
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Scheme 1. Synthesis of N-substituted SAHA analogs (2a–e).
Inhibitor screening with lysates
values of >500 mM (Table 1). While the N-substituent
significantly reduced the potency of the analogs, these
studies with HeLa cell lysates do not assess their selectivities.
HDAC inhibitory activities of analogs 2a–e were measured
using the Fluor de LysTM in vitro fluorescence activity assay
(Biomol Inc.) and the HDAC activity from HeLa cell lysates. All
analogs showed significantly reduced potency compared to
SAHA (Table 1). Pentyl variant 2b displayed the most potent
activity with a 153 mM IC50 value, which is 1700-fold reduced
compared to the parent SAHA (0.090 mM, Table 1). In contrast,
the methyl variant 2a was the least potent with a 794 mM IC50
value, which is >8800-fold reduced compared to SAHA. The
benzyl analog 2c demonstrated an intermediate IC50 of
293 mM. Unfortunately, the homobenzyl and biphenyl var-
iants 2d and 2e displayed low solubility at high concen-
trations, prohibiting IC50 determination. The percentage
activity remaining at the highest concentration of analog
tested (250 mM) suggested that 2d and 2e demonstrate IC50
Inhibitor screening with HDAC1, 3, and 6
To assess selectivity, the analogs were screened against three
individual HDAC isoforms, HDAC1, 3, and 6. HDAC1 and 3
were included because compound 1 was able to discriminate
between them in prior work [25, 26]. HDAC6 was also tested
to assess class II selectivity. The analogs were initially screened
against the isoforms at a single concentration of 125 mM
(Fig. 2). The compounds containing aliphatic substituents (2a,
methyl; and 2b, pentyl) displayed little to no isoform
selectivity, similar to SAHA [6]. Likewise, the N-homobenzyl
analog 2d also showed roughly similar potency against the
isoforms. In contrast, the N-benzyl 2c and N-biphenyl 2e
Table 1. HDAC inhibitory potency of 2a–e and SAHA against HeLa cell lysates.
Compounds
R
IC50 (mM) or % activity remaininga)
SAHA
2a
2b
2c
2d
H
0.090 ꢁ 0.004
794 ꢁ 41
Methyl
Pentyl
Benzyl
153 ꢁ 14
293 ꢁ 32
Homobenzyl
Biphenyl
80 ꢁ 2% activity at 250 mMb)
2e
101 ꢁ 1% activity with 250 mMb)
a) Values are the mean of three independent trials with standard error (Supporting Information Tables S1–S4 and Figs. S1–S4).
b) Solubility problems prevented testing at concentrations above 250 mM. Note that assays with the methyl variant 2a at 250 mM
showed 72 ꢁ 5% remaining activity (Supporting Information Table S5).
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233 ꢁ 40 mM. Unfortunately, due to insolubility at high
concentrations, IC50 values for HDAC3 and HDAC6 could
not be determined. However, at the highest concentration
possible (250 mM), no inhibitory activity was observed with
either HDAC3 or HDAC6 (Supporting Information Table S11),
suggesting a preference for HDAC1.
Docking studies
Docking studies were performed to rationalize the lower
potency and HDAC1 preference of the N-modified SAHA
analogs. SAHA, along with the N-pentyl 2b and N-biphenyl 2e
analogs, were docked into the HDAC1 crystal structure [13].
̊
SAHA displayed five interactions (1.1–3.8 A distances) with the
bound Zn2þ metal and nearby amino acids (H140, H141, and
Y303, Fig. 3A, see residues in blue). In contrast, the N-pentyl
2b variant maintained only three of these interactions (with
Zn2þ, H141, and Y303, Fig. 3B), while the N-biphenyl 2e analog
contained only two (with Zn2þ and Y303, Fig. 3C). The loss of
hydrogen bonding by the hydroxamic acid amine likely
accounts for the fewer stabilizing interactions with the
analogs. In addition, the orientation of the hydroxamic acid
is altered by the N-modification. Specifically, SAHA positions
the carbonyl adjacent to Y303, the amine near H141, and
the hydroxyl next to H140. Due to the N-modification, the
hydroxyl amine orientation is flipped with N-pentyl 2b
positioning the hydroxyl near H141. Likewise, the N-biphenyl
analog adopts an alternative pose with the carbonyl
interacting with Zn2þ and the hydroxyl interacting with
Y303. The docking experiments point toward fewer inter-
actions between the hydroxamic acid and the active site, likely
Figure 2. HDAC inhibitory activities of the N-modified SAHA
analogs were measured at 125 mM against HDAC1, 3, and 6.
SAHA was tested at 125 nM concentration. Mean percentage
deacetylase activity remaining of three to five independent
trials with standard error is shown (Supporting Information
Table S6).
variants displayed preferential HDAC1 inhibition, similar
to compound 1. Among these two analogs, N-biphenyl
SAHA 2e demonstrated the greatest preference, with
58 ꢁ 2% activity remaining with HDAC1 but statistically
insignificant inhibition observed with both HDAC3 and
HDAC6. The single concentration selectivity screen points to
N-biphenyl 2e as a preferential HDAC1 inhibitor, similar to
compound 1.
̊
due to flipping of the N-modification into the 14-A cavity and
loss of hydrogen bonding, which account for the reduced
potency.
In addition to the loss of bonding interactions, another
significant observation from the docking studies is the
̊
positioning of the N-modification within the 14-A internal
Based on the observation that the benzyl 2c and biphenyl
2e variants displayed HDAC1 preference at 125 mM concen-
tration, IC50 values were determined (Table 2). As expected [6],
cavity. The narrowest section of the cavity is created by M30,
R34, and L139 (Fig. 3A and Supporting Information Fig. S9A,
see resides in purple). While the pentyl group of 2b is
positioned up to this constricted point in the cavity (Fig. 3B),
the biphenyl group of 2e extends beyond the narrow opening
(Fig. 3C). Therefore, the reduced potency of N-biphenyl SAHA
2e may also be due to the narrowing of the cavity near M30,
R34, and L139 to constrict binding (Supporting Information
the pan-inhibitor SAHA displayed less than
a 1.5-fold
preference for any HDAC isoform tested [29]. The benzyl
variant 2c displayed a modest preference for HDAC1 versus
HDAC3 (2.5-fold). The N-biphenyl variant 2e displayed
preferential inhibition for HDAC1, with an IC50 of
Table 2. IC50 values for SAHA, N-benzyl 2c, and N-biphenyl 2e against HDAC1, 3, and 6.a)
HDAC1
HDAC3
HDAC6
SAHA
2c
2e
0.096 ꢁ 0.016 mM
177 ꢁ 21 mM
0.146 ꢁ 0.012 mM
440 ꢁ 23 mM
105 ꢁ 2%b)
0.074 ꢁ 0.009 mM
287 ꢁ 9 mM
233 ꢁ 40 mM
108 ꢁ 3%b)
a) Values are the means of at least three independent trials with standard error (Supporting Information Tables S7–S10 and
Figs. S5–S8).
b) Deacetylase activity remaining at 250 mM concentration of inhibitor is shown (Supporting Information Table S11) because
solubility issues prevented IC50 value determination.
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Figure 3. Docking of SAHA (A), N-pentyl SAHA 2b (B), and N-biphenyl SAHA 2e (C) into the HDAC1 crystal structure (PBD 4BKX). The
HDAC structure is represented as blue mesh, the Zn2þ metal as a blue orb, and the inhibitor and amino acids as ball and sticks. The
atoms of the inhibitor are color-coded (C ¼ green/white; O ¼ red; N ¼ blue, H ¼ white).
Fig. S9A). Consistent with the extent and orientation of
interactions, the energies of the inhibitor/HDAC1 complexes
for SAHA, N-pentyl SAHA 2b, and N-biphenyl SAHA 2e
were ꢂ5.46, ꢂ4.25, and ꢂ3.28 kcal/mole, respectively. The
energies are consistent with the experimental data indicating
that SAHA is the most potent compound, whereas the
N-biphenyl 2e analog is the least potent (Table 1).
that S113 is only partially responsible for the potency of
compound 1 with HDAC1 [21].
An alternative hypothesis explaining the HDAC1 prefer-
ences of compound 1 and N-biphenyl SAHA 2e emerges when
̊
considering the narrowest point in the 14-A internal cavities
of HDAC1 and HDAC3. HDAC3 maintains a considerably more
̊
constricted 14-A internal cavity than HDAC1 (compare
The docking studies were further analyzed to explain the
enhanced preference of 2e for HDAC1 compared to SAHA
and 2b (Table 2). Prior docking analysis with compound 1
suggested that selectivity for HDAC1 compared to HDAC3 was
Fig. 4A to Fig. 3A). The residues M24, R28, and L133 of
HDAC3 appear to block the cavity and may restrict access to
bulky N-modified inhibitors. Docking of N-biphenyl 2e into
the HDAC3 crystal structure produced no poses that were
consistent with the expected metal/hydroxamic binding
interaction. Instead, the biphenyl group was positioned up
to the narrowest section of the cavity, near M24, R28, and
L133 of HDAC3 (Fig. 4C and Supporting Information Fig. S9B).
In this case, the biphenyl group is unable to extend beyond
̊
a result of congestion in the 14-A cavity due to a tyrosine in
HDAC3; HDAC1 contains serine at the same position, which
allows the cavity to accommodate bulky aromatic groups [26].
Docking of SAHA into the HDAC3 structure revealed that
̊
Y107 is positioned relatively distant to the 14-A cavity
̊
(Fig. 4A) [13, 14]. Likewise, S113 in the HDAC1 crystal structure
is also located adjacent to the cavity (Supporting Information
Fig. S10). In addition, previous mutagenesis studies indicated
the constricted region to access the 14-A internal cavity of
HDAC3, as was seen with HDAC1 (Fig. 3C and Supporting
Information Fig. S9A). As a result of the blocked cavity, the
Figure 4. Docking of SAHA (A), N-pentyl SAHA 2b (B), and N-biphenyl SAHA 2e (C) into the HDAC3 crystal structure (PBD 4A69). The
HDAC structure is represented as blue mesh, the Zn2þ metal as a gray orb, and the inhibitor and amino acids as ball and sticks.
See Fig. 3 for color coding.
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hydroxamic acid is positioned at the outside edge of the active
site channel, unable to interact with the metal ion. Therefore,
docking suggests that the poor potency observed with 2e and
HDAC3 is due to restricted access of the N-biphenyl to the
“Iron-free” glassware was prepared by soaking in a 6 M HCl
acid bath overnight. Tetrahydrofuran (THF) was freshly
distilled before use from sodium benzophenone ketyl,
dichloromethane (CH2Cl2) was freshly distilled before use
from CaH2, and triethylamine was freshly distilled before use
̊
14-A internal cavity, which prevents favorable metal/hydroxa-
̊
mic acid interactions. In contrast, the straight chain N-pentyl
analog 2b is positioned up to the constriction point (Fig. 4B),
similar to HDAC1 (Fig. 3B), which allows effective binding and
better potency. In total, the docking studies are consistent
from CaH2. Flash chromatography was carried out using 60-A,
230–400 mesh silica gel. All organic reagents were used as
purchased from Acros Organics, Fisher Scientific, or Sigma–
Aldrich. NMR spectra were taken on either a Varian Unity
300 MHz, Varian L900 400 MHz, or a Varian 500 MHz. IR
spectra were taken on a Jasco FT/IR – 4100. HRMS data were
obtained on either a Waters LCT Premier XE ESI-LC–MS TOF or
a Waters GCT EI-TOF. Syringe filters used were 0.22 mm
Millipore Millex Syringe Drive Filter Unit, PES express.
̊
with accessibility of the 14-A cavity to large aromatic groups
as a significant factor leading to the HDAC1 preference of
inhibitors bearing substituents on the metal binding group,
including compound 1 and N-biphenyl SAHA 2e.
Please also see the Supporting Information for the InChI
codes and selected biological activities of the compounds 2a–e.
Discussion
N-modified SAHA analogs (2a–e) displayed significantly
reduced potency compared to the parent SAHA. Interestingly,
the benzyl and pentyl substituents are tolerated to a greater
extent than any of the other N-substituted analogs. Docking
studies are consistent with the pentyl groups accessing the
Preparation of suberic anhydride (4)
Suberic anhydride (4) was synthesized as described [32].
Accordingly, suberic acid (5.0 g, 28.8 mmol) was added to
acetic anhydride (10 mL) and refluxed for 1 h. The mixture was
then evaporated to remove excess acetic anhydride. The
residue was recrystallized several times from acetonitrile to
give suberic anhydride (2.0 g, 45%). All spectra were identical
with those described in the literature.
̊
14-A internal cavities of HDAC1 and HDAC3 (Figs. 3B and 4B).
However, the additional interactions in the cavity were
unable to overcome the lost hydrogen bonding due to the
presence of the N-modification. The results suggest that any
group, regardless of size, incorporated directly on the
hydroxamic acid will result in decreased inhibitory activity
compared to the unsubstituted analog. These studies are
consistent with prior work reporting reduced potencies of
Preparation of 8-oxo-8-(phenylamino)octanoic acid (5)
8-Oxo-8-(phenylamino)octanoic acid (5) was synthesized as
described [32]. Suberic anhydride (1.0 g, 6.4 mmol) was
dissolved in THF (10 mL). Aniline (0.6 mL, 6.4 mmol) was then
added and the solution was allowed to stir for 30 min. The
mixture was diluted with water (100 mL) and the formed solid
was filtered and recrystallized from water to give 8-oxo-8-
(phenylamino)octanoic acid (1.6 g, 55%). All spectra were
identical with those described in the literature.
HDAC inhibitors as
a result of N-methylation of the
hydroxamic acid group [30, 31].
Although the N-modified SAHA analogs showed reduced
potency compared to SAHA, one compound displayed HDAC1
preference. The N-biphenyl variant 2e showed an IC50 of
233 mM with HDAC1, while not inhibiting HDAC3 or 6 at the
highest concentration allowed (250 mM). Docking analysis
with the HDAC1 and HDAC3 crystal structures suggests that
Preparation of tert-butyl benzyloxycarbamate (7)
tert-Butyl
benzyloxycarbamate
was
synthesized
as
̊
that accessibility to the 14-A internal cavity is differentially
described [33]. Accordingly, to a flask containing O-benzylhy-
droxylamine hydrochloride (1.0 g, 6.23 mmol) was added
dioxane (7 mL). Di-tert-butyl dicarbonate (1.33 mL, 6.23 mmol)
was added, followed by a 1 M solution of NaHCO3 (6.23 mL,
6.23 mmol). Vigorous evolution of gas was observed. The
solution was allowed to stir overnight. The mixture was then
evaporated to remove dioxane. To the residue was added a
1 M solution of citric acid (until pH was ꢃ4). The mixture was
then extracted with dichloromethane (3 ꢄ 25 mL). The organic
layers were pooled, dried over magnesium sulfate, filtered,
and evaporated to afford tert-butyl benzyloxycarbamate
(1.4 g, 99%). All spectra were identical with those described in
the literature.
restricted, leading to preferential binding to HDAC1 over
HDAC3. Therefore, the combined experimental and compu-
tational analyses of N-modified SAHA analogs further
validate the concept of creating isoform-selective HDAC
̊
inhibitors by positioning aromatic substituents in the 14-A
internal cavity. These studies guide future inhibitor design by
suggesting that additional substituted metal binding groups
can be created to take advantage of the altered cavity
accessibility of the HDAC isoforms.
Experimental
Chemistry
General methods
Typical procedure for the generation of tert-butyl
benzyloxy(alkyl)carbamate (8)
Unless otherwise noted, all manipulations were carried out
tert-Butyl benzyloxy(alkyl)carbamate was synthesized as
under inert argon atmosphere in flame-dried glassware.
described [33]. Accordingly, to a flask containing 1 equiv.
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of tert-butyl benzyloxycarbamate (7) was added THF (to make
a 0.1 M solution) followed by 1.25 equiv. of sodium hydride
(60% dispersion on mineral oil). Vigorous evolution of gas was
observed as the flask was allowed to stir for 30 min. The alkyl
halide (1.25 equiv.) was then added and the mixture was
heated overnight. The mixture was filtered through celite,
evaporated to an oil, and purified using flash silica gel
chromatography (5% ethyl acetate/hexanes) to afford tert-
butyl benzyloxy(alkyl)carbamate as a clear oil.
with sat. NaHCO3 (1 ꢄ volume to organic layer) to neutralize
the remaining TFA. The aqueous layer was then washed with
dichloromethane (2 ꢄ volume to aqueous layer). The organic
layers were pooled, dried over magnesium sulfate, filtered,
and evaporated to give the deprotected amine. To the flask
containing the amine was then added acetonitrile (0.1 M
solution based on acid 5), 1 equiv. of 8-oxo-8-(phenylamino)-
octanoic acid (5), 1.5 equiv. of TBTU, and 1.25 equiv.
of diisopropylethylamine, successively. The mixture was
allowed to stir overnight. The reaction was then quenched
with 1 M HCl aqueous solution and extracted with ethyl
acetate (3 ꢄ volume to aqueous layer). The organic layers
were pooled, dried over magnesium sulfate, filtered, and
evaporated to an oil. Flash silica gel chromatography (5%
methanol, 45% dichloromethane, 50% hexanes) afforded
N1-(benzyloxy)-N1-alkyl-N8-phenyloctanediamide (10) as a
clear oil.
Preparation of tert-butyl benzyloxy(methyl)carbamate
(8a)
Methyl iodide (0.0704 mL, 1.13 mmol) gave 8a (0.2105 g, 98%)
1H NMR (500 MHz, CDCl3): d 1.50 (s, 9H), 3.05 (s, 3H), 4.85 (s,
2H), 7.30–7.40 (m, 5H); 13C NMR (125 MHz, CDCl3): d 28.5, 37.0,
77.0, 81.0, 128.5, 129.5, 136.0, 157.0.
Preparation of tert-butyl benzyloxy(pentyl)carbamate (8b)
Bromopentane (0.28 mL, 2.2 mmol) gave 8b (0.1095 g, 23%).
1H NMR (500 MHz, CDCl3): d 0.90 (t, 3H), 1.30 (m, 4H), 1.50 (s,
9H), 1.65 (t, 2H), 3.40 (t, 2H), 4.85 (s, 2H), 7.35–7.41 (m, 5H);
13C NMR (125 MHz, CDCl3): d 14.2, 22.2, 27.0, 28.5, 29.2, 50.0,
77.0, 81.5, 128.5, 129.5, 136.0, 157.0; IR: 2958, 2932, 2871,
1702, 1455, 1367, 1276, 1150, 747, 699 cmꢂ1. HRMS (ESI-LC–
MS, m/z); found: [MþNa], 316.1896, calcd. for C27H27NO3Na,
316.1889.
Preparation of N1-(benzyloxy)-N1-methyl-N8-
phenyloctanediamide (10a)
tert-Butyl
benzyloxy(methyl)carbamate
(8a)
(0.20 g,
0.84 mmol) gave 10a (0.1469 g, 71%). 1H NMR (500 MHz,
CDCl3): d 1.30 (m, 4H), 1.60 (m, 2H), 1.70 (m, 2H), 2.30–2.38
(m, 4H), 3.20 (s, 3H), 4.8 (s, 2H), 7.1 (t, 1H), 7.3 (m, 2H), 7.40 (m,
5H), 7.55 (d, 2H), 7.85 (bs, 1H); 13C NMR (125 MHz, CDCl3): d
24.5, 25.5, 29.0, 29.2, 32.5, 34.0, 37.5, 76.5, 120.0, 124.0,
129–129.8 (m), 135.0, 139.0, 171.5, 176.0; IR: 3309, 2930, 2857,
1660, 1599, 1541, 1498, 1440, 1176, 977, 753, 698 cmꢂ1; HRMS
(ESI-LC–MS, m/z); found: [M], 368.2099, calcd. for C22H28N2O3,
368.2100.
Preparation of tert-butyl benzyloxy(phenethyl)carbamate
(8d)
(2-Bromoethyl)benzene (0.19 mL, 1.35 mmol) gave 8d
(0.2367 g, 80%). 1H NMR (500 MHz, CDCl3): d 1.43 (s, 9H),
2.90 (t, 2H), 3.60 (t, 2H), 4.80 (s, 2H), 7.20–7.50 (m, 10H);
13C NMR (125 MHz, CDCl3): d 28.4, 33.5, 41.8, 77.0, 81.8, 126.5,
129.0, 129.4, 129.5, 130.0, 136.0, 139.0, 157.0; IR: 2976, 2932,
Preparation of N1-(benzyloxy)-N1-pentyl-N8-
phenyloctanediamide (10b)
tert-Butyl benzyloxy(pentyl)carbamate (8b) (0.1 g, 0.34 mmol)
gave 10b (0.1217 g, 84%). 1H NMR (500 MHz, CDCl3): d 0.90 (t,
3H), 1.25–1.40 (m, 8H), 1.60 (m, 4H), 1.72 (t, 2H), 2.35 (t, 2H),
2.40 (m, 2H), 3.65 (m, 2H), 4.80 (s, 2H), 7.1 (t, 1H), 7.3 (t, 2H),
7.40 (m, 5H), 7.60 (d, 2H), 7.9 (bs, 1H); 13C NMR (125 MHz,
CDCl3): d 14.0, 22.5, 24.8, 25.5, 27.0, 29.0, 29.1, 29.2, 32.4, 37.8,
45.8, 77.0, 120.0, 124.0, 129.0–129.4 (m), 135.0, 138.5, 171.0,
175.0; IR: 3308, 2931, 2858, 1661, 1599, 1541, 1498, 1440, 753,
697 cmꢂ1. HRMS (ESI-LC–MS, m/z); found: [MþH], 425.2799,
calcd. for C26H37N2O3, 425.2804, found: [MþNa], 447.2611,
calcd. for C26H36N2O3, 447.2624.
1699, 1366, 1157, 746, 698 cmꢂ1
.
Preparation of tert-butyl benzyloxy(biphenyl-4-ylmethyl)-
carbamate (8e)
4-(Bromomethyl)biphenyl (0.2793 g, 1.13 mmol) gave 8e
(0.3268 g, 93%). 1H NMR (500 MHz, CDCl3): d 1.50 (s, 9H),
4.61 (s, 2H), 4.7 (s, 2H), 7.32 (m, 6H), 7.45 (m, 4H), 7.60 (dd, 4H);
13C NMR (125 MHz, CDCl3): d 29.0, 54.0, 77.0, 82.0, 127.5, 127.7,
129.0, 129.2, 129.4, 129.8, 135.8, 136.1, 141.0, 141.2, 157.0;
IR: 2975, 2930, 1698, 1366, 1159, 1082, 757, 697 cmꢂ1; HRMS
(ESI-LC–MS, m/z); found: [MþNa], 412.1886, calcd. for
C
25H27NO3Na, 412.1889.
Preparation of N1-(benzyloxy)-N1-phenethyl-N8-
phenyloctanediamide (10d)
Typical procedure for the generation of N1-(benzyloxy)-
N1-alkyl-N8-phenyloctanediamide (10)
tert-Butyl benzyloxy(phenethyl)carbamate (8d) (0.2367 g,
0.72 mmol) gave 10d (0.1422 g, 65%). 1H NMR (500 MHz,
CDCl3): d 1.30 (m, 2H), 1.35 (m, 2H), 1.58 (m, 2H), 1.70 (m, 2H),
2.30–2.40 (m, 4H), 2.91 (t, 2H), 3.90 (m, 2H), 4.80 (s, 2H), 7.10 (t,
1H), 7.20 (m, 4H), 7.30 (m, 4H), 7.40 (m, 4H), 7.55 (d, 2H), 7.80
(bs, 1H); 13C NMR (125 MHz, CDCl3): d 24.5, 25.5, 29.0, 29.1,
32.5, 33.5, 37.8, 47.8, 77.0, 120.0, 124.0, 127.0, 129 (m), 135.0,
138.8, 139.0, 172.0, 175.0; IR: 3311, 2931, 1660, 1599, 1540,
1497, 1440, 751, 698 cmꢂ1; HRMS (ESI-LC–MS, m/z); found: [M],
To a flask containing 1.5 equiv. of tert-butyl benzyloxy(alkyl)-
carbamate (8) was added a 50% TFA in dichloromethane
solution (0.16 M solution based on 8) and the mixture was
allowed to stir for 10 min (until completeness visualized by
TLC, Rf ¼ 0 compared with Rf ¼ 0.5 for 8, 5% ethyl acetate in
hexanes). The mixture was then evaporated to remove TFA.
The residue was dissolved in dichloromethane and washed
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459.2648, calcd. for C29H35N2O3, 459.2648, found: [MþNa],
carbon. The flask was vacuum purged several times with
argon and then hydrogen before incubation under hydrogen
for 2 h with stirring (until completeness visualized by TLC,
Rf ¼ 0 compared with Rf ¼ 0.5 for 9, 30% ethyl acetate in
hexanes). The mixture was filtered and then solvents
evaporated to afforded N1-hydroxy-N1-alkyl-N8-phenyl-
octanediamide (2) as a white solid.
481.2458, calcd. for C29H35N2O3Na, 481.2458.
Preparation of N1-(benzyloxy)-N1-(biphenyl-4-ylmethyl)-
N8-phenyloctanediamide (10e)
tert-Butyl benzyloxy(biphenyl-4-ylmethyl)carbamate (8e)
(0.32 g, 0.82 mmol) gave 10e (0.1724 g, 40%). 1H NMR
(500 MHz, CDCl3): d 1.30 (m, 4H), 1.65 (m, 2H), 1.70 (m, 2H),
2.30 (t, 2H), 2.45 (t, 2H), 4.80 (s, 2H), 4.85 (s, 2H), 7.10 (t, 1H),
7.30–7.45 (m, 12H), 7.5–7.60 (m, 6H); 13C NMR (125 MHz,
CDCl3): d 24.5, 25.5, 29.0, 29.1, 32.8, 37.8, 50.0, 77.0, 120.0,
124.0, 127.2, 127.4, 129.0 (m), 135.0, 136.0, 138.5, 140.8, 141.0,
171.8; IR: 3308, 2930, 1660, 1599, 1541, 1498, 1488, 1440, 910,
Preparation of N1-hydroxy-N1-methyl-N8-
phenyloctanediamide (2a)
N1-(Benzyloxy)-N1-methyl-N8-phenyloctanediamide
(10a)
(0.1400 g, 0.39 mmol) gave 2a (0.1070 g, 98%). 1H NMR
(500 MHz, CD3OD): d 1.40 (m, 4H), 1.60 (m, 2H), 1.70 (m,
2H), 2.35 (t, 2H), 2.45 (t, 2H), 3.19 (s, 3H), 7.10 (t, 1H), 7.30
(t, 2H), 7.50 (d, 2H); 13C NMR (125 MHz, CD3OD): d 25.0, 25.8,
29.0, 29.1, 31.9, 35.0, 36.8, 120.0, 124.0, 128.5, 139.0, 173.5,
175.0; HRMS (EI-TOF, m/z); found: [M], 278.1635, calcd. for
755, 698 cmꢂ1
543.2628, calcd. for C34H36N2O3Na, 543.2624.
;
HRMS (ESI-LC–MS, m/z); found: [MþNa],
Preparation of N,O-dibenzylhydroxylamine (9)
N,O-Dibenzylhydroxylamine was synthesized as described
previously [34]. Accordingly, O-benzylhydroxylamine (2 g,
9.4 mmol) hydrochloride was dissolved in DMF (50 mL).
Potassium carbonate (5.2 g, 37.5 mmol) was added to the
flask followed by benzyl bromide (1.5 mL, 12.5 mmol). The
mixture was stirred overnight. The reaction was quenched
with water (30 mL) and extracted with CH2Cl2 (4 ꢄ 20 mL). The
organic layers were pooled and evaporated to an oil. To
remove residual DMF, the oil was dissolved in 20 mL of diethyl
ether and extracted with water (3 ꢄ 20 mL). The ethereal layer
was dried over magnesium sulfate, filtered, and evaporated
to an oil. Flash silica gel chromatography (5% ethyl acetate in
hexanes) afforded 1.83 g of a clear oil (67%). All spectra were
identical with those described in the literature.
C15H22N2O3, 278.1630.
Preparation of N1-pentyl-N1-hydroxy-N8-
phenyloctanediamide (2b)
N1-(Benzyloxy)-N1-pentyl-N8-phenyloctanediamide
(10b)
(0.12 g, 0.28 mmol) gave 2b (0.0860 g, 88%). 1H NMR
(500 MHz, CD3OD): d 0.90 (t, 3H), 1.25–1.43 (m, 8H), 1.63 (m,
4H), 1.70 (m, 2H), 2.39 (t, 2H), 2.49 (t, 2H), 3.60 (t, 2H), 7.10 (t,
1H), 7.30 (t, 2H), 7.55 (d, 2H); 13C NMR (125 MHz, CD3OD): d
13.0, 22.2, 24.8, 25.8, 26.1, 28.78, 28.9, 29.0, 32.0, 36.9,
47.8, 120.0, 124.0, 128.8, 138.9, 173.5, 174.8; IR: 3310, 3190,
2930, 1663, 1617, 1600, 1573, 1549, 1510, 1500, 1465, 1258,
1214, 1173, 1033, 751, 725, 690 cmꢂ1; HRMS (ESI-LC–MS, m/z);
found: [MþH], 335.2345, calcd. for C19H31N2O3, 335.2335,
found: [MþNa], 357.2151, calcd. for C19H31N2O3Na, 357.2154.
Preparation of N1-benzyl-N1-(benzyloxy)-N8-
phenyloctanediamide (10c)
Preparation of N1-benzyl-N1-hydroxy-N8-
To
a
flask containing N,O-dibenzylhydroxylamine (9)
phenyloctanediamide (2c)
(0.1920 g, 0.9 mmol) was added acetonitrile (6 mL), 8-oxo-8-
(phenylamino)octanoic acid (5) (0.15 g, 0.6 mmol), TBTU
(0.28 g, 0.9 mmol), and diisopropylethylamine (0.21 mL,
1.2 mmol), successively. The general procedure was followed
to produce 10, except that the chromatography solvent was
30–40% ethyl acetate/hexanes, which afforded N1-benzyl-N1-
(benzyloxy)-N8-phenyloctanediamide (10c) (0.1754 g, 63%).
1H NMR (500 MHz, CDCl3): d 1.30 (m, 4H), 1.60 (m, 2H), 1.70 (m,
2H), 2.30 (t, 2H), 2.42 (t, 2H), 4.75 (s, 2H), 4.81 (s, 2H), 7.10 (t,
1H), 7.25–7.38 (m, 12H), 7.55 (d, 2H), 7.60 (bs, 1H); 13C NMR
(125 MHz, CDCl3): d 24.2, 25.8, 29.0, 29.1, 32.3, 37.8, 50.5, 77.0,
120.0, 124.0, 128.0, 128.72, 128.8, 128.9, 129.15, 129.42, 135.0,
137.0, 138.5, 171.8; IR: 3315, 2932, 1661, 1599, 1541, 1497,
1440, 1308, 911, 753, 698 cmꢂ1; HRMS (EI-TOF, m/z); found:
[M], 444.2424, calcd. for C28H32N2O3, 444.2413.
N1-Benzyl-N1-(benzyloxy)-N8-phenyloctanediamide
(10c)
(0.17 g, 0.38 mmol) gave 2c (0.1223 g, 91%). 1H NMR
(500 MHz, CD3OD): d 1.40 (m, 4H), 1.65 (m, 4H), 2.35 (t, 2H),
2.50 (t, 2H), 4.85 (s, 2H), 7.10 (t, 1H), 7.30 (m, 7H), 7.55 (d, 2H);
13C NMR (125 MHz, CD3OD) 0.0, 124.0, 127.5, 128.1, 128.2,
128.5, 137.0, 139.0, 173.5, 175.0; IR: 3304, 3136, 2937, 1660,
1596, 1531, 1497, 1468, 1443, 1204, 1181, 754, 720, 696 cmꢂ1
HRMS (EI-TOF, m/z); found: [M], 354.1957, calcd. for
21H26N2O3, 354.1943.
;
C
Preparation of N1-hydroxy-N1-phenethyl-N8-
phenyloctanediamide (2d)
N1-(Benzyloxy)-N1-phenethyl-N8-phenyloctanediamide (10d)
(0.14 g, 0.31 mmol) gave 2d (0.0791 g, 69%). 1H NMR
(500 MHz, CD3OD): d 1.31–1.40 (m, 4H), 1.55 (m, 2H), 1.70
(m, 2H), 2.35 (t, 2H), 2.45 (t, 2H), 2.90 (t, 2H), 3.80 (t, 2H), 7.10
(t, 1H), 7.15–7.30 (m, 7H), 7.55 (d, 2H); 13C NMR (125 MHz,
CD3OD): d 24.5, 25.5, 28.8, 29.0, 32.0, 32.8, 36.7, 49.0, 120.0,
124.0, 126.0, 128.2, 128.3, 128.5, 139.0, 174.0, 175.0; IR: 3167,
Typical procedure for the generation of N1-hydroxy-N1-
alkyl-N8-phenyloctanediamide (2)
To an acid washed flask containing 1 equiv. of N1-(benzyloxy)-
N1-alkyl-N8-phenyloctanediamide (10) was added ethyl ace-
tate (0.1 M solution) and 0.1 equiv. of 10% palladium on
2932, 2849, 1649, 1600, 1499, 1463, 1413, 1191, 753, 691 cmꢂ1
HRMS (ESI-LC–MS, m/z); found: [MþH], 369.2195, calcd. for
.
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N-Modified SAHA Analogs
Archiv der Pharmazie
C22H29N2O3, 369.2178, found: [MþNa], 391.2015, calcd. for
Figs S1–S8). Data with SAHA (lysates and isoforms) were
previously reported, but included here for comparison [29].
C
22H28N2O3Na, 391.1998.
Preparation of N1-(biphenyl-4-methyl)-N1-hydroxy-N8-
phenyloctanediamide (2e)
Docking studies
Crystal structures for HDAC1 and HDAC3 were downloaded
from the RCSB protein data bank (HDAC1: 4BKX and HDAC3:
4A69). PyMOL (Schrodinger, LLC) was used to delete the MTA1
corepressor chain, acetate, potassium, and sulfate ions in the
HDAC1 crystal structure. In the case of the HDAC3 crystal
structure, the water molecules, chain A, deacetylase-activa-
tion-domain (DAD) (from the SMRT corepressor), glycerol,
D-myo-inositol-1,4,5,6-tetrakisphosphate molecules, acetate,
potassium, and sulfate ions were deleted. AutoDockTools-
1.5.4 program [37, 38] was used to add all hydrogen atoms,
modify histidine protonation (H140 and H141 residues for
HDAC1, His134 and His135 for HDAC3) by adding only HD1,
compute Gasteiger charges, and merge all nonpolar hydro-
gen, followed by generation of the pdbqt output file. The
charge of the zinc atom was manually changed from zero to
N1-(Benzyloxy)-N1-(biphenyl-4-ylmethyl)-N8-phenyloctane-
diamide (10e) (0.1700 g, 0.33 mmol) gave 2e (0.0986 g, 69%).
1H NMR (500 MHz, DMSO): d 1.30 (m, 4H), 1.55 (m, 4H), 2.25 (t,
2H), 2.40 (t, 2H), 4.70 (s, 2H), 7.00 (t, 1H), 7.22 (m, 3H), 7.30 (m,
2H), 7.42 (t, 2H), 7.60 (m, 6H), 9.85 (bs, 1H); 13C NMR (125 MHz,
DMSO): d 25.0, 25.8, 29.0, 29.1, 32.3, 37.0, 51.5, 120.0, 123.5,
127.0, 128.0, 129.0, 129.1, 129.3, 137.0, 139.8, 140.0, 140.5,
172.0, 174.0; HRMS (ESI-LC–MS, m/z); found: [MþH], 431.2342,
calcd. for C27H31N2O3, 431.2335, found: [MþNa], 453.2159,
calcd. for C27H30N2O3Na, 453.2154.
HDAC activity assay
HDAC activity was measured using the Fluor de LysTM assay
(Biomol), as previously reported [35, 36]. To measure global
HDAC inhibition, 0.5 mL of HeLa lysates (ꢃ4 mg of total
protein) diluted up to 24 mL with HDAC assay buffer (50 mM
Tris, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) was
incubated without (1 mL of DMSO) or with small molecule
inhibitor (1 mL of 50ꢄ stock solution in DMSO, final
̊
þ2. A grid box with a spacing of 0.375 A, size of 56 ꢄ 42 ꢄ 38,
and coordinates for the center of the grid box (ꢂ48.000,
18.000, ꢂ3.750) were used for HDAC1, while the values for
HDAC3 were 58 ꢄ 58 ꢄ 54 and (8.166, 76.663, 21.318). The
map type was set by choosing the ligand and then AutoGrid
4.2 was used to pre-calculate and generate the grid maps files
required for the docking calculations. All the docked
compounds were drawn in ChemBioDraw Ultra and Chem
3D Pro was used to run MM2 job for energy minimization.
Then AutoDockTools-1.5.4 program was used to add hydro-
gens, compute Gasteiger charges, merge nonpolar hydro-
gens, choose torsions, and generate the pdbqt files. All acyclic
bonds were made rotatable except amide bonds. AutoDock
4.2 program [38] was used to perform the docking calcu-
lations using a genetic algorithm. The generated pdbqt file
for the enzyme was set as a rigid macromolecule and the
genetic algorithm search parameters were set to 100 GA runs
for each ligand with a population size of 150, a maximum
number of 2.5 ꢄ 105 energy evaluations, a maximum number
of 2.7 ꢄ 104 generations, a mutation rate of 0.2, and a
crossover rate of 0.8. The docking parameters were set to
default. All the output DLG files were converted to pdbqt
format and the results were visualized in PyMOL. The lowest
energy pose consistent with metal binding is discussed in
the text.
concentrations of inhibitors indicated in Fig.
2 and
Supporting Information Tables S1–S11) for 30 min at
30°C with shaking. To perform the isoform selective studies,
HDAC1 (0.2 mg, specific activity ¼ 42.5 pmol/min/mg),
HDAC3 (0.05 mg, specific activity ¼ 249 pmol/min/mg)
or HDAC6 (0.25 mg, specific activity ¼ 257 pmol/min/mg)
purchased from BPS Biosciences was used. After the pre-
incubation period, Fluor de LysTM substrate (25 mL) in HDAC
assay buffer was added (to give a final concentration of
100 mM for HeLa lysate, 50 mM for HDAC1, or 25 mM for
HDAC3 and 6 in a total reaction volume of 50 mL). The
reaction mixture was incubated at 30°C for 45 min with
shaking. Fluor de LysTM developer (2.5 mL of 20ꢄ stock
solution diluted up to 50 mL in HDAC assay buffer) was
added (to give a 100 mL total volume) and incubated with
shaking for 5 min at room temperature. The fluorescence
intensity was determined (excitation at 360 nm and
emission at 465 nm) using a Geniosplus Fluorimeter (Tecan).
For each trial, a reaction without HDAC activity was used
to assess the background. The background fluorescence
without HDAC activity was subtracted from the signal
observed with enzyme. Percentage deacetylase activity was
calculated by dividing the background corrected fluores-
cence units of small molecule-treated reactions with that of
untreated reactions (set at 100%) and multiplying by 100.
At least three independent trials were performed with the
mean and standard error reported. IC50 values were
obtained by plotting the percentage deacetylase activity
versus small molecule concentration and fitting the data
to a sigmoidal dose–response curve (y ¼ 100/(1 þ (x/IC50)n)
using KaleidaGraph software (Supporting Information
Research reported in this publication was supported by the
National Institute of General Medical Sciences of the National
Institutes of Health under Award Number R01GM067657. The
content is solely the responsibility of the authors and does
not necessarily represent the official views of the National
Institutes of Health. We also thank the Wayne State University
for funding, and G. Padige and J. Piechocki for technical
support.
The authors have declared no conflict of interest.
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A. V. Bieliauskas et al.
Archiv der Pharmazie
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