Structural requirements of HDAC inhibitors: SAHA analogs functionalized adjacent to the hydroxamic acid

Article abstract of DOI:10.1016/j.bmcl.2007.01.117

Inhibitors of histone deacetylase (HDAC) proteins such as suberoylanilide hydroxamic acid (SAHA) have emerged as effective therapeutic anti-cancer agents. To better understand the structural requirements of HDAC inhibitors, a small molecule library with a

Full text of DOI:10.1016/j.bmcl.2007.01.117

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2216–2219
Structural requirements of HDAC inhibitors: SAHA analogs
functionalized adjacent to the hydroxamic acid
Anton V. Bieliauskas, Sujith V. W. Weerasinghe and Mary Kay H. Pflum*
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Received 29 December 2006; revised 22 January 2007; accepted 22 January 2007
Available online 8 February 2007
Abstract—Inhibitors of histone deacetylase (HDAC) proteins such as suberoylanilide hydroxamic acid (SAHA) have emerged as
effective therapeutic anti-cancer agents. To better understand the structural requirements of HDAC inhibitors, a small molecule
library with a variety of substituents attached adjacent to the metal binding hydroxamic acid of SAHA was synthesized. The pres-
ence of a substituent adjacent to the hydroxamic acid led to an 800- to 5000-fold decrease in inhibition compared to SAHA. The
observed results have implications for drug design, suggesting that HDAC inhibitors with substituents near the metal binding
moiety will have inhibitory activities in the micromolar rather than nanomolar range.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Suberoylanilide hydroxamic acid (SAHA, Vorinostat,
ZolinzaTM) recently gained FDA approval for the treat-
ment of advanced cutaneous T-cell lymphoma (CTCL).1
SAHA is an inhibitor ofhistone deacetylase (HDAC) pro-
teins, which are linked to a variety of cancers.2 While
SAHA is the first HDAC inhibitor (HDACi) to meet
FDA approval, several other small molecules that inhibit
HDAC proteins are currently in clinical trials for cancer
treatment.3 Distinguishing characteristics of HDAC
inhibitors include a metal binding moiety, a carbon linker,
and a capping group (Fig. 1). Based on crystallographic
Figure 1. Structures of select HDAC inhibitors.
analyses, the capping group is solvent-exposed and
interacts with amino acids near the entrance of the active
site, while the metal binding moiety resides in the protein
interior and complexes the metal ion involved in cataly-
proic acid (Fig. 1), contain benzamide and carboxylate
sis.4–6 The linker serves to position the capping and
metal binding groups appropriately for high-affinity
interactions with proteins. With a modular framework
and application toward cancer treatment, HDAC inhibi-
tors are viable targets for future drug design.
metal binding moieties, respectively,9,10 and display
IC50 values of 2 and 400 lM.11,12 The reduced inhibitory
activities compared to SAHA (110–370 nM IC50)13,14
are partially explained by the presence of the benzamide
or carboxylic acid group.7,15,16 Interestingly, MS-275
displays modest preference toward select proteins within
the 11-membered HDAC family.17 Selective HDAC
inhibitors would aid in elucidating the role of each indi-
vidual HDAC protein in cancer and have the potential
to be better drugs.18 However, strictly selective HDAC
inhibitors have yet to be discovered.
Previous HDACi design has emphasized modification of
the capping group and the metal binding moiety. In the
case of the metal binding moiety, SAHA contains a
hydroxamic acid while other inhibitors contain thiols,
epoxides, carboxylates, or benzamides.7,8 For example,
two HDAC inhibitors in clinical trials, MS-275 and val-
In addition to altering the metal binding moiety toward
HDACi design, the hydrocarbon linker has been diversi-
fied, focusing on altering chain length, creating points of
unsaturation along the chain, and including an aryl or
Keywords: Histone deacetylase; HDAC inhibitor; HDACi; SAHA.
*
Corresponding author. Tel.: +1 313 577 1515; fax: +1 313 577
8822; e-mail: pflum@chem.wayne.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.117

A. V. Bieliauskas et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2216–2219
2217
cyclohexyl ring within the chain.19–22 However, few
studies have examined the impact of substituents on the
linker chain. Recently, small molecules bearing substitu-
ents on the linker adjacent to the capping group were
shown to not only display nanomolar inhibition, but also
modest isoform selectivity.15 In contrast, the incorpora-
tion of substituents on the linker adjacent to the metal
binding moiety has a variable influence on inhibitory
activity. Two reports noted that a methyl substituent near
the hydroxamic acid of hydroxamate based libraries led to
120- to 170-fold decreased HDAC inhibition compared to
the unsubstituted analog, although the potency was in the
micromolar range.23,24 In addition, the short chain fatty
acid valproic acid contains an propyl group adjacent to
the carboxylate and inhibits in the micromolar range. In
contrast, small molecules bearing an intra-chain aryl
group near the hydroxamic acids display nanomolar
HDAC inhibition.20,21 For example, MS-275 bears an
aryl group adjacent to the benzamide group and displays
potent HDAC inhibition.11 Therefore, the influence of
substituents on the linker adjacent to the hydroxamic acid
remains unclear. The structural requirements of HDAC
inhibitors, particularly on the linker chain, are an
interesting and relatively unexplored area of study.
Modification of known HDAC inhibitors is necessary to
identify the structural factors influencing inhibitor
potency and provide insight for designing new inhibitors.
Scheme 1. Reagents and conditions for the racemic synthesis of
compounds 1a–g: (i) PhNH2, AlMe3, THF, 98%; (ii) MsCl, TEA,
CH2Cl2, 99%; (iii) a—NaH, dimethylmalonate, THF; b—mesylate
from (ii), THF, reflux, 90%; (iv) NaH, RX, THF, reflux, 33–98%; (v)
a—LiCl, H2O, DMSO, reflux; b—NaOH, MeOH, reflux, 67–83%; (vi)
Ethyl chloroformate, N-methylmorpholine, NH2OH, MeOH, 10–24%;
(vii) CDI, TEA, NH2OBn, THF, reflux, 75–91%; (viii) H2, Pd–C,
MeOH, 48%; (ix) BCl3, CH2Cl2, 84–85%.
activity assay kit.29 IC50 values were obtained by fitting
the data to a sigmoidal dose–response curve (Fig. 2).
The structure–activity relationship of compounds 1a–g
is summarized in Table 1. All of the SAHA analogs
inhibited HDAC activity in the micromolar range. The
butyl variant 1d was the most potent analog displaying
an IC50 of 72 lM, while the ethyl variant 1b displayed
the weakest inhibitory activity of 449 lM. Interestingly,
the analogs containing the smallest (1a-methyl) or the
largest (1g-benzyl) substituents displayed HDAC inhib-
itory activities between those of 1d and 1b. In addition,
the propargyl analog 1f inhibited to almost the same
level as the butyl analog 1d (87 and 72 lM, respectively).
The results indicate steric considerations alone cannot
predict the inhibitory activities of the C2-substituted
analogs.
To probe the structural requirements of HDAC inhibi-
tors, analogs of SAHA with substituents adjacent to
the hydroxamic acid were tested. Specifically, we synthe-
sized a small library of SAHA analogues (1) bearing a
variety of hydrophobic substituents at the C2 position.25
Hydrophobic substituents were selected because crystal-
lographic analysis of HDAC proteins indicates that the
active site residues surrounding the linker chain are
hydrophobic.4–6 The synthetic route for the small mole-
cule library is outlined in Scheme 1. e-Caprolactone (2)
was opened with aniline to give anilide alcohol 3. The
alcohol was activated prior to incubation with the anion
of dimethyl malonate to give diester 4. The diester was
deprotonated and treated with a variety of alkyl halides
to afford compounds 5a–g. Krapcho type decarboxyl-
ation26 and subsequent saponification gave compounds
6a–g. Carboxylic acids 6a–d were converted directly to
the hydroxamic acids 1a–d with ethyl chloroformate
and a hydroxylamine solution. However, the low yields
encouraged utilization of a second strategy where
hydroxamic acids 1e–g were synthesized via the benzyl
protected hydroxamic acids 7e–g followed by deprotec-
27
tion by either H2/Pd–C for compound 1g, or BCl3
for unsaturated compounds 1e and 1f. Yields after the
two-step hydroxamic acid installation/benzyl deprotec-
tion were superior to direct conversion (38% and 64%
compared to 10–24%). Although reported for benzyl
ethers, we note that use of BCl3 to remove a benzyl
group on a hydroxamic acid is unestablished to the best
of our knowledge.28 A more thorough exploration of the
scope and limitations of the BCl3 deprotection reaction
is currently under investigation.
HDAC inhibitory activities of the SAHA analogs were
measured using the Fluor de LyseTM in vitro fluorescence
Figure 2. Dose–response curves of SAHA analogues 1a–g from three
independent trials with error bars indicating standard error.

2218
A. V. Bieliauskas et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2216–2219
Table 1. HDAC inhibition by compounds 1a–g, SAHA, and MS-275
Pflum for advice, and Emily Aubie, Fabiola Bittencourt,
Paulina Karwowska-Desaulniers, and Kevin Kells for
comments.
a
Compounds
R
IC50 (lM)
SAHA
MS-275
1a
0.090 ( 0.004)
3.2 ( 0.1)
134 ( 6)
449 ( 17)
154 ( 7)
72 ( 6)
Methyl
Ethyl
1b
1c
References and notes
n-Propyl
n-Butyl
Allyl
1d
1e
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While the SAHA analogs displayed inhibitory activities
in the micromolar range, all had significantly decreased
inhibitory activities when compared to those of SAHA
(90 nM) or MS-275 (3.2 lM). The most potent butyl
variant 1d demonstrated 800- and 20-fold decreased
activity compared to SAHA and MS-275, respectively.
The weakest ethyl variant 1b displayed 5000- and 128-
fold decreased inhibition compared to SAHA and
MS-275. The results suggest that any group, regardless
of size, incorporated adjacent to the hydroxamic acid
will result in decreased inhibitory activity compared to
the unsubstituted analog.
8. Suzuki, T.; Nagano, Y.; Kouketsu, A.; Matsuura, A.;
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Several HDAC inhibitors maintain similar nanomolar
potency compared to SAHA yet contain a ring within
the carbon linker adjacent to the metal binding moie-
ty.20,21 The fact that SAHA analogs with substituents
at the C2 position display micromolar IC50 values indi-
cates that only modest steric bulk near the hydroxamic
acid is tolerated for nanomolar inhibitory activity.
Therefore, the results suggest that the steric environment
near the hydroxamic acid in the HDAC active site is sig-
nificantly confined. The results have implications for
anti-cancer drug design, predicting that HDAC inhibi-
tors with substituents near the hydroxamic acid will
have inhibitory activities in the micromolar range.
9. Atmaca, A.; Maurer, A.; Heinzel, T.; Gottlicher, M.;
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16. Park, J. H.; Jung, Y.; Kim, T. Y.; Kim, S. G.; Jong, H. S.;
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SAHA analogs with substituents adjacent to the cap-
ping group were potent nanomolar inhibitors.15 In con-
trast, SAHA analogs with substituents adjacent to the
hydroxamic acid demonstrated micromolar IC50 values.
The combined data suggest that substituents are
tolerated along the linker chain but potency diminishes
when positioned near the metal binding moiety.
Because modest isoform selectivity has been reported
with HDAC inhibitors bearing substituents along the
linker,15,17
a systematic assessment of substituent
tolerance along the linker chain will guide future
HDACi design. The effect of incorporating substituents
at additional positions along the linker chain is currently
under investigation.
18. Karagiannis, T. C.; El-Osta, A. Leukemia 2006, 21, 61.
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We thank the National Institute of Health (GM067657)
and Wayne State University for funding, Derek A.

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C17H24N2O3, 304.1787. Compound 1f: 85% yield from
7f. Mp 138–140 ꢁC; 1H NMR(500 MHz, CD3OD) d
(ppm): 1.25–1.5 (m, 4H), 1.6 (m, 2H), 1.7 (m, 2H), 2.2–
2.45 (m, 6H), 7.1 (t, 1H), 7.3 (t, 2H), 7.5 (t, 2H); 13C NMR
(125 MHz, CD3OD) d (ppm): 22.0, 25.0, 27.0, 29.0, 32.0,
37.0, 43.5, 70.3, 81.3, 120.0, 124.0, 129.0, 139.0, 173.0,
175.0; HRMS (ESI-LC–MS, m/z); found: [M], 302.1640,
calcd for C17H22N2O3, 302.1630. Compound 1g: 48% yield
from 7g. Mp 138–140 ꢁC; 1H NMR (500 MHz, CD3OD) d
(ppm): 1.3 (m, 4H), 1.5 (m, 2H), 1.7 (m, 2H), 2.35 (m, 2H),
2.65 (m, 1H), 2.7 (m, 1H), 2.9 (m, 1H), 7.1 (t, 1H), 7.17 (m,
3H), 7.2 (t, 2H), 7.3 (t, 2H), 7.5 (t, 2H); 13C NMR
(125 MHz, CD3OD) d (ppm): 25.0, 27.0, 29.0, 37.0, 39.0,
46.0, 120.0, 124.0, 126.0, 128.0, 128.5, 129.0, 139.0, 140.0,
173.0, 174.0; HRMS (ESI-LC–MS, m/z); found: [M],
354.1957, calcd for C21H26N2O3, 354.1943.
23. Woo, S. H.; Frechette, S.; Khalil, E. A.; Bouchain, G.;
Vaisburg, A.; Bernstein, N.; Moradei, O.; Leit, S.; Allan,
M.; Fournel, M.; Trachy-Bourget, M. C.; Li, Z.; Bes-
terman, J. M.; Delorme, D. J. Med. Chem. 2002, 45, 2877.
24. Lavoie, R.; Bouchain, G.; Frechette, S.; Woo, S. H.;
Khalil, E. A.; Leit, S.; Fournel, M.; Yan, P. T.; Trachy-
Bourget, M.-C.; Beaulieu, C.; Li, Z.; Besterman, J.;
Delorme, D. Bioorg. Med. Chem. Lett. 2001, 11, 2847.
25. Hydroxamic acid characterization. No spurious peaks
were observed in the NMR spectra of the synthesized
compounds. Compound 1a: 33% yield from 6a. Mp 128–
130 ꢁC; 1H NMR (500 MHz, CD3OD) d (ppm): 1.3 (d,
3H), 1.35–1.4 (m, 6H), 1.6–1.7 (m, 4H), 2.2 (m, 1H), 2.4 (t,
2H), 7.1 (t, 1H), 7.3 (t, 2H), 7.5 (t, 2H); 13C NMR
(125 MHz, CD3OD) d (ppm): 17.0, 25.0, 27.0, 29.0, 34.0,
37.0, 38.0, 120.0, 124.0, 129.0, 139.0, 173.0, 175.0; HRMS
(ESI-LC–MS, m/z); found: [M], 278.1640, calcd for
C15H22N2O3, 278.1630. Compound 1b: 14% yield from
6b. Mp 121–123 ꢁC; 1H NMR (500 MHz, CD3OD) d
(ppm): 0.9 (t, 3H), 1.25–1.5 (m, 6H), 1.6 (m, 2H), 1.7 (m,
2H), 1.95 (m, 1H), 2.4 (t, 2H), 7.1 (t, 1H), 7.3 (t, 2H), 7.5
(t, 2H); 13C NMR (125 MHz, CD3OD) d (ppm): 11.0,
25.8, 25.9, 27.1, 29.0, 32.3, 36.7, 45.0, 120.0, 124.0, 129.0,
139.0, 173.0, 175.0; HRMS (ESI-LC–MS, m/z); found:
[MÀH2O], 274.1687, calcd for C16H22N2O2, 274.1681.
Compound 1c: 10% yield from 6c. Mp 164–166 ꢁC; 1H
NMR (500 MHz, CD3OD) d (ppm): 0.9 (t, 3H), 1.25–1.5
(m, 8H), 1.6 (m, 2H), 1.7 (m, 2H), 2.1 (m, 1H), 2.35 (t,
2H), 7.1 (t, 1H), 7.3 (t, 2H), 7.5 (t, 2H); 13C NMR
(125 MHz, CD3OD) d (ppm): 13.0, 20.7, 25.9, 27.0, 29.0,
33.0, 35.0, 37.0, 43.7, 120.0, 124.0, 129.0, 139.0, 173.5,
175.0; HRMS (ESI-LC–MS, m/z); found: [MÀH2O],
288.1851, calcd for C17H24N2O2, 288.1838. Compound
1d: 24% yield from 6d. Mp 153–156 ꢁC; 1H NMR
(500 MHz, CD3OD) d (ppm): 0.9 (t, 3H), 1.2–1.5 (m,
10H), 1.6 (m, 2H), 1.7 (m, 2H), 2.05 (m, 1H), 2.35 (t, 2H),
7.1 (t, 1H), 7.3 (t, 2H), 7.5 (t, 2H); 13C NMR (125 MHz,
CD3OD) d (ppm): 13.0, 22.5, 25.5, 27.0, 29.0, 30.0, 32.4,
32.5, 37.0, 44.0, 120.0, 124.0, 129.0, 139.0, 173.0, 175.0;
HRMS (ESI-LC–MS, m/z); found: [MÀH2O], 302.2000,
calcd for C18H26N2O2, 302.1994. Compound 1e: 84% yield
from 7e. Mp 143–146 ꢁC; 1H NMR (500 MHz, CD3OD) d
(ppm): 1.25–1.5 (m, 4H), 1.6 (m, 2H), 1.7 (m, 2H), 2.1 (m,
2H), 2.3 (m, 1H), 2.35 (t, 2H), 5.0 (q, 2H), 5.7 (m, 1H), 7.1
(t, 1H), 7.3 (t, 2H), 7.5 (t, 2H); 13C NMR (125 MHz,
CD3OD) d (ppm): 25.5, 27.0, 29.0, 32.0, 36.5, 37.0, 44.0,
116.0, 120.05, 124.0, 129.0, 136.0, 139.0, 174.0; HRMS
(ESI-LC–MS, m/z); found: [M], 304.1797, calcd for
26. Krapcho, A. P.; Glynn, G. A.; Grenon, B. J. Tetrahedron
Lett. 1967, 8, 215.
27. In a representative procedure, 0.12 g (0.3 mmol) of O-
benzyl protected hydroxamic acid 7e was dissolved in
3 ml THF and cooled to 0 ꢁC via an ice bath. 1.5 ml
(5 equiv) of a 1 M BCl3 solution in CH2Cl2 was then
added dropwise. The ice bath was removed after 5 min
and the reaction mixture was stirred for an additional 3 h
(complete by TLC analysis) at room temperature. The
mixture was then quenched with 1 M HCl and extracted
with ethyl acetate (3· 10 ml). The organic layers were
pooled, dried over magnesium sulfate, and evaporated
onto silica gel. Flash chromatography (10% MeOH/
CH2Cl2) with acid-washed (deferrated) silica gel afforded
the hydroxamic acid 1e in 84–85% yield. For a repre-
sentative procedure for obtaining deferrated silica gel, see
Guo, H.; Naser, S. A.; Ghobrial, G.; Phanstiel, O.
J. Med. Chem. 2002, 45, 2056.
28. Farr, R. A.; Bey, P.; Sunkara, P. S.; Lippert, B. J. J. Med.
Chem. 1989, 32, 1879, provides an example of an
unsuccessful hydroxamic acid O-benzyl deprotection with
BCl3.
29. The HDAC activity was measured using Fluor de LyseTM
activity assay (Biomol). Briefly, HeLa lysates (25 lL) were
incubated with or without the small molecule inhibitor for
30 min at 30 ꢁC with shaking. Fluor de Lys substrate
(25 lL, 100 lM) was added and the reaction mixture was
incubated at 37 ꢁC for 45 min with shaking. Fluor de Lys
developer (50 lL of 1·) was added and incubated with
shaking for 5 min. The fluorescence intensity was deter-
mined at 465 nm using a Genios Fluorimeter (Tecan). The
deacetylase activity was determined by dividing the
fluorescence intensity of the reaction in the presence of
SAHA analog with the intensity in the absence of
inhibitor. At least three determinations were used to
calculate the mean and standard error in Figure 2 and
Table 1.