Structure-activity relationships of aryloxyalkanoic acid hydroxyamides as potent inhibitors of histone deacetylase

Article abstract of DOI:10.1016/j.bmcl.2006.09.085

Syntheses of aryloxyalkanoic acid hydroxyamides are described, all of which are potent inhibitors of histone deacetylase, some being more potent in vitro than trichostatin A (IC₅₀ = 3 nM). Variation of the substituents on the benzene ring as we

Full text of DOI:10.1016/j.bmcl.2006.09.085

Bioorganic & Medicinal Chemistry Letters 17 (2007) 136–141
Structure–activity relationships of aryloxyalkanoic acid
hydroxyamides as potent inhibitors of histone deacetylase
a
a
a
Charles M. Marson,a, Thevaki Mahadevan, Jon Dines, Stephane Sengmany,
*
´
James M. Morrell,a John P. Alao,b, Simon P. Joel,c David M. Vigushinb,z and
R. Charles Coombesb
aDepartment of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H OAJ, UK
bDepartment of Cancer Medicine, 8th Floor MRC Cyclotron Building, Imperial College,
Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK
cCentre for Medical Oncology, Institute of Cancer, St. Bartholomew’s Hospital, West Smithfield, London EC1A 7BE, UK
Received 3 August 2006; revised 26 September 2006; accepted 26 September 2006
Available online 30 September 2006
Abstract—Syntheses of aryloxyalkanoic acid hydroxyamides are described, all of which are potent inhibitors of histone deacetylase,
some being more potent in vitro than trichostatin A (IC50 = 3 nM). Variation of the substituents on the benzene ring as well as
fusion of a second ring have marked effects on potency, in vitro IC50 values down to 1 nM being obtained.
Ó 2006 Elsevier Ltd. All rights reserved.
Inhibitors of histone deacetylase (HDAC) enzymes have
recently gained prominence as an emerging class of anti-
cancer agents.1,2 HDAC enzymes catalyze the deacetyla-
tion of e-amino groups of lysine residues in the N-termi-
nal tails of core histones in the nucleosome, resulting in
protonated e-amino lysine residues that interact strongly
with DNA, leading to a less accessible, compacted chro-
matin structure.3 In this way, HDACs can act as repres-
sors of gene transcription by altering the accessibility of
transcription factors to DNA.4,5 The inappropriate
recruitment of HDAC enzymes by oncogenic proteins
may alter gene expression in favor of arrested differenti-
ation, and/or excessive proliferation.6 Conversely, inhib-
itors of HDACs are able to relieve transcriptional
Several HDAC inhibitors including suberoylanilide
hydroxamic acid (SAHA),11 (Fig. 1) NVP-LAQ824,12
MS-275,13 and the cyclodepsipeptide FK-22814 have en-
tered clinical trials. Those and the natural product tri-
chostatin A (TSA)15 induce differentiation in cancer
cell lines and suppress cell proliferation. The efficacy
of such HDAC inhibitors is generally considered to de-
pend upon the presence of three major features: (1) a ter-
minal group (hydroxamic acid in the present class of
novel inhibitors) that can bind to zinc in the active site
of HDAC enzymes and which is connected to (2) a link-
er unit, residing in the channel, which in turn is cova-
lently bonded to (3) an end moiety (or capping group)
that principally occupies the external entrance to the
channel of the enzyme. Such a model is in agreement
with structures of histone deacetylase-like protein
(HDLP) co-crystallized with either TSA or SAHA.16
repression of certain genes including the cyclin-depen-
6,7
dent kinase inhibitor protein p21WAF1/CIP1
,
and such
HDAC inhibitors typically inhibit cancer cell prolifera-
tion by induction of cell cycle arrest, differentiation
and/or apoptosis.8–10
As part of our anti-cancer program centered on therapy
using small molecules, we have used the framework of
trichostatin A to design novel HDAC inhibitors.17
While both TSA and SAHA contain carbonyl groups
(keto and amide, respectively) as part of the linker, we
(CMM and SPJ) have also shown that HDAC inhibito-
ry potency can be maintained by a sulfoxide, or merely a
thioether of type 1 in the linker unit.18 Compounds con-
taining those groups showed moderate inhibition in the
enzyme assay employed,18 suggesting that an oxygen
Keywords: Histone deacetylase; Enzyme inhibitors; Hydroxamic acids;
Aryloxyalkanoic acid derivatives.
*
Corresponding author. Tel.: +44 20 76794712; fax: +44 20
76797463; e-mail: c.m.marson@ucl.ac.uk
Present address: Department of Molecular Biology, Lundberg
Laboratory, Go¨teborg University, Box 462, S-405 30 Go¨teborg,
zSweden.
Deceased.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.085

C. M. Marson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 136–141
137
O
O
O
O
OH
O
S
OH
R2
R1
O
OH
N
N
N
H
n
n
H
H
N
R
Trichostatin A
1
2
NH
N
H
N
OH
N
H
O
O
OH
N
H
SAHA
HO
NVP-LAQ824
Figure 1.
atom interposed between an aromatic ring and an alkyl
chain could also be of interest and perhaps furnish more
potent inhibitors of HDAC than the thioether ana-
logues.19 Here, we report that aromatic ether-linked
compounds, principally of type 2, are indeed compatible
with very potent HDAC inhibition, with IC50 values
extending down to 1 nM.
ation of a suitable phenol with an x-bromo ester 4 in the
presence of Cs2CO3 (Scheme 1).20 Since an N-substitut-
ed p-aminophenyl moiety was a common feature of
many of the inhibitors 2 and their precursors, selective
O-alkylation was a requirement of the routes. Moreover,
a variety of substituents at nitrogen were to be investi-
gated; both requirements could be met by O-alkylation
of a p-nitrophenol 3 to give the ethers 5 followed by
reduction to the p-amino derivatives 6. Such amines 6
proved satisfactory in a variety of reductive alkylations
Esters of aryloxyalkanoic acids were conveniently pre-
pared by the Williamson ether synthesis involving alkyl-
O
O
O
R1
R1
R1
OH
O
O
b
a
Br
OEt
OEt
OEt
+
O2N
O
O2N
H2N
3
5
6
4
O
R1
R1
2
O
O
c
d
3
OEt
NHOH
R2
R2
N
N
R3
7
8
R3
Scheme 1. Reagents and conditions: (a) Cs2CO3, DMF, 16 h, reflux; (b) H2, 5% Pd–C, EtOH, 3h; (c) R2CHO, (R3CHO), NaCNBH3, (2 equiv),
ZnCl2, MeOH; (d) 50% aq NH2OH (9 equiv), KOH (1.7 equiv) in MeOH–THF. Compound key: a, R1 = H, R2 = R3 = Me; b, R1 = H, R2 = Me,
R3 = p-ClC6H4CH2; c, R1 = H, R2 = Me, R3 = 4-pyridylmethyl; d, R1 = R3 = H, R2 = 5-indolylmethyl; e, R1 = R3 = H, R2 = 4-(3-hydroxy-5-
hydroxymethyl-2-methyl)pyridylmethyl; f, R1 = 3-fluoro, R2 = H, R3 = 4-(3-hydroxy-5-hydroxymethyl-2-methyl)pyridylmethyl; g, R1 = H, R2 = 3-
indolylpropionyl, R3 = 4-pyridylmethyl.
O
O
O
O
O
O
O
O
a
b
b
OEt
OEt
NHOH
NHOH
OEt
O
O
O O
2
2
n
S
S
R1
N
R1
N
6a n=1
n=2
10 n=3
H2N
11
13
12
14
9
H
H
O
O
R2
R2
2
3
O
O
c or d
6, 9 or 10
n
n
R1
N
H
R1
N
H
Scheme 2. Reagents and conditions: (a) R1SO2Cl, Et3N, CH2Cl2, 2 h; (b) 50% aq NH2OH (6 equiv), KOH (1 equiv) in MeOH–THF; (c) R1CO2H,
iso-butyl chloroformate, N-methylmorpholine, THF; (d) R1CO2H, 1-hydroxybenzotriazole, 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide, N-
methylmorpholine, THF. Compound key: for 11 and 12: R1 = p-MeOC6H4; for 13 and 14: a, R1 = 3-indolylacetyl, R2 = H, n = 1; b, R1 = 3-
indolylacetyl, R2 = H, n = 2; c, R1 = 3-indolylacetyl, R2 = H, n = 3; d, R1 = 3-indolylethyl, R2 = H, n = 2; e, R1 = 3-indolylacetyl, R2 = 3-fluoro,
n = 2; f, R1 = 3-indolylacetyl, R2 = 2-fluoro, n = 2, R1 = 3-indolylpropionyl, R2 = H, n = 2.
O
O
O
OH
O
O
X
X
X
b
Br
a
OEt
NHOH
OEt
2
2
+
2
15
16
18
17
Scheme 3. Reagents and conditions: (a) Cs2CO3, DMF, 16 h, reflux; (b) 50% aq NH2OH (9 equiv), KOH (1.7 equiv) in MeOH–THF. Compound
key: a, X@CH@CH; b, X@NH.

Table 1. In vitro inhibition of histone deacetylase21,22
Compound Structure
HDAC IC50 (nM) Compound Structure
HDAC IC50 (nM)
H
N
Me2N
O
H
N
8a
18.8 6.0
14a
50 21
OH
O
X
N
H
O
N
H
O
O
O
OH
O
Cl
H
N
Me
N
O
8b
O
2.0 1.0
14b (X=O)
14d (X=H2)
12.8 5.2 (14b)
6.2 3.5 (14d)
OH
N
H
OH
N
H
O
N
H
H
N
N
Me
N
O
H
8c
8d
15.3 6.2
14c
14e
22.4 9.0
O
N
N
H
OH
OH
O
N
O
H
H
F
N
H
N
H
N
O
O
14.3 5.7
23.5 9.0
O
O
O
OH
OH
N
H
O
OH
N
H
O
N
H
OH
H
N
N
F
H
N
8e
8f
22.0 12.0
14f
13.1 5.0
O
O
N
H
O
N
H
OH
OH
OH
O
O
N
H
H
N
OH
N
F
H
N
H
N
70 28
14g
16.0 5.5
O
O
OH
O
N
OH
N
H
H
N
H
N
O
8g
12
16.4 8.0
18a
18b
0.9 0.3
N
OH
OH
O
O
N
H
O
OH
O
N
H
MeO
H
H
N
N
O
S
O
22.1 13.3
3.6 1.8
O O
O
N
OH
O
N
H
H
In vitro inhibition data for A-161906 (IC50 = 9 nM)19 and trichostatin A (IC50 = 3 nM).21

C. M. Marson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 136–141
139
(Scheme 1): a one-pot N,N-dimethylation using aqueous
37% formaldehyde (6 equiv) afforded 7a. Reductive
monoalkylations also proceeded well, affording the sec-
ondary amines 7b–f. Treatment of those esters with
aqueous 50% hydroxylamine afforded the corresponding
hydroxamic acids 8a–f. A similar procedure afforded,
after acylation with 3-indolylpropionic acid using meth-
od c (Scheme 2) the ester 7g which with aqueous 50%
hydroxylamine gave the hydroxamic acid 8g.
Table 1 shows in vitro IC50 values of various com-
pounds described above as measured against deacetyla-
tion of histone H4.21,22 Preliminary modeling studies
indicated that the binding energy of a saturated chain
ether 8 is greater than the corresponding a,b,c,d-unsat-
urated system despite the conformational constraints
imposed by a diene unit. Accordingly, this work con-
cerns only the more readily obtained saturated com-
pounds 8 which have the additional advantage of not
being capable of undergoing conjugate addition. In
designing inhibitors, consideration was also given to
the need for a branch point such that the aromatic por-
tion can adopt an obtuse angle with respect to the main
linker chain in the tunnel of the enzyme (as in Fig. 2).
Given the lengths of the linker units in TSA and SAHA,
the heptanoic acid hydroxyamides were expected to be
of suitable length; at least in the case of series 14a,
14b, and 14c, the heptanoic acid hydroxyamide 14b
was the most potent. Those indolylacetamide derivatives
were initially selected partly because of ease of synthesis,
but also because in the Novartis LAQ series12 (Fig. 1),
the presence of an indole ring was found to be necessary
for good cellular anti-proliferation values.12a Introduc-
tion of a fluoro substituent (amides 14e and 14f) showed
no appreciable effect for the 2-fluoro derivative, and
somewhat lower potency for the 3-fluoro derivative,
although all compounds 14d–f were potent inhibitors
The amino ether 6a underwent sulfonylation to give 11.
Acylation of the appropriate amine 6a, 9, or 10 fur-
nished esters 13a,13b,13e, and 13f using iso-butyl chlo-
roformate in the presence of N-methylmorpholine,
esters 13c, 13d, and 13g using EDCI-HOBt.20 (The sub-
sequent reduction of the amide with NaBH4 (1 mol e-
quiv) in the presence of acetic acid (1 equiv) at 45 °C
for 17 h furnished 13d). Treatment of those esters with
aqueous 50% hydroxylamine afforded the corresponding
hydroxamic acids 12 and 14a–g.
Fused ring phenols 15a and 15b were conveniently alkyl-
ated with ethyl 7-bromoheptanoate (16) to give the
corresponding ethers 17a and 17b.20 Treatment of
those esters with aqueous 50% hydroxylamine afforded
the corresponding hydroxamic acids 18a and 18b
(Scheme 3).
Figure 2. Molecular modeling of the binding of ligands 18a (magenta), 18b (yellow), and TSA (turquoise) to HDAC1 using AutoDock3.023 and
viewed in PyMOL. Upper picture: cap region of the enzyme; lower picture: side-on view of the three ligands and their coordination to zinc (brown
sphere) with depiction of residues in and near the catalytic site.23

140
C. M. Marson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 136–141
of the HDAC cell extract. The longer indolylpropiona-
mide group of 14g afforded comparable activity to the
indolylacetamide analog 14b, implying that the addi-
tional flexibility arising from an extra methylene group
did not confer, overall, more beneficial contacts to the
cap region. Perhaps surprisingly, the additional hydro-
phobic region afforded by the 4-pyridylmethyl group
present in 8g did not confer a greater observed potency
than that of 14g.
MCF-7 cell lines (IC50 = 7 lM for 14d and 5 lM for
18b).
Acknowledgments
Financial support from the EPSRC (to TM and JM)
and from BBSRC (to SS on Grant GR31/B18148) is
gratefully acknowledged. We are also grateful for sup-
port by the Mandeville Trust (studentship to JD).
Possessing more conformational flexibility than the cor-
responding amides, the secondary amines 8d and 14d
were prepared and tested. Both were potent inhibitors
of HDAC, the latter somewhat more so, possibly owing
to the less rigid chain which could enable more ready
presentation of the indole ring to binding regions on
the protein (compare also 14b with 14d). Among the sec-
ondary amines, the polar 4-pyridylmethyl derivatives 8e
and 8f, bearing polar substituents, were examined;
whereas hydroxamic acid 8e is of comparable potency
to both the less polar compounds in the phenoxy series
(e.g., 8a, 8d and especially 8c), the 3-fluoro derivative 8f
was much weaker. This marked difference, which is not
found in the analogous pair 14e and 14f, might be
accounted for in terms of diminished capability of the
secondary amino nitrogen atom of 8f to engage in
hydrogen bonding.
References and notes
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The derivative 8a showed that a simple N,N-dimethyla-
mino substituent (corresponding to the cap region of
TSA) is sufficient to give good potency. However, exten-
sion of the hydrophobic area of the cap group can great-
ly increase potency; this can be achieved by means of a
flexible (aminomethyl) unit, as in 8b, or by ring fusion,
as in 18a and 18b. In all three cases, significantly greater
potency than that of A-161906 was achieved, showing
the potential for further improvements in such aryloxy-
alkanoic acid hydroxyamides. Modeling of TSA in
HDAC1 using AutoDock3.023 gave a binding of TSA
(Fig. 2) consistent with previous modeling24 and with
its location in HDLP as determined by crystallogra-
phy.16 The planar rings of 18a and 18b contribute to
the potency, probably by preferentially occupying the
relatively planar and hydrophobic portion of the cap re-
gion of the enzyme as shown in Figure 2.23
17. Marson, C. M.; Serradji, N.; Rioja, A. S.; Gastaud, S. P.;
Alao, J. P.; Coombes, R. C.; Vigushin, D. M. Bioorg.
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19. A 40-cyanobiphenylyl ether designated A-161906 from an
Abbott compound library has been reported to be an
HDAC inhibitor, but details were scant: (a) Glaser, K. B.;
Li, J.; Pease, L. J.; Staver, M. J.; Marcotte, P. A.; Guo, J.;
Frey, R. R.; Garland, R. B.; Heyman, H. R.; Wada, C. K.;
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683; (b) Frey, R. R.; Wada, C. K.; Garland, R. B.; Curtin,
M. L.; Li, J.; Pease, L. J.; Glaser, K. B.; Marcotte, P. A.;
Bouska, J. J.; Murphy, S. S.; Davidsen, S. K. Bioorg. Med.
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In conclusion, the aryloxyalkanoic acid hydroxyamides
herein described are some of the most potent HDAC
inhibitors known in relation to their structural simplici-
ty. The potencies of aryloxyalkanoic acid hydroxya-
mides indicate that neither a carbonyl group (as
present in trichostatin A and SAHA) nor a rigid (alkyl-
ene) chain is essential for low nanomolar HDAC enzyme
inhibition to be achieved. General features of structure–
activity relationships can be discerned for this series of
inhibitors, and a mode of docking similar to that accept-
ed for TSA16,24 is proposed for some of the compounds
herein described. These aryloxy inhibitors of histone
deacetylase may prove to be of superior in vivo stability
compared with the well-studied amidic HDAC inhibi-
tors that can be cleaved by peptidases. Additionally,
hydroxamic acids 14d and 18b inhibited proliferation
of HeLa (IC50 = 7 lM for both compounds) and
20. Yields for compounds in Scheme 1: 5a (93%), 5b (85%); 6a
(98%), 6b (83%); 5e (74%), 5f (79%); 6e (95%), 6f (89%); 7a

C. M. Marson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 136–141
141
(69%), 7b (68%), 7c (72%), 7d (67%), 7e (77%), 7f (70%); 8a
(38%), 8b (31%), 8c (32%), 8d (31%), 8e (59%), 8f (40%); 7g
(85%); 8g (81%). Yields for compounds in Scheme 2: 11
(63%); 13a (85%), 13b (78%), 13e (83%), 13f (94%); 13c
(82%), 13d (78%), 13g (79%); 13d (53%); 12 (64%); 14a
(59%), 14b (36%), 14c (42%), 14d (69%), 14e (77%), 14f
(28%); 14g (39%). Yields for compounds in Scheme 3: 17a
(79%), 17b (60%); 18a (24%), 18b (66%).
quantification by liquid scintillation counting. The sub-
strate was a synthetic peptide corresponding to the N-
terminal of the released product ([3H]acetic acid) with
ethyl acetate and quantification by liquid scintillation
counting. Each test compound was assayed in duplicate
whilst positive control (trichostatin A) and negative
control samples were assayed in triplicate. Further details
are given in note 16 of Ref. 17.
21. Vigushin, D. M.; Aboagye, E.; Buluwela, L.; Coombes, C.
Clin. Cancer Res. 2001, 7, 971.
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R.; Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput.
Chem. 1998, 19, 1639 http://www.scripps.edu/pub/olson-
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pub/olson-web/doc/autodock/tools.html. For use of PyMOL
and viewing of protein-ligand structures see: http://www.
pymol.org/funding.html.
22. Histone deacetylase activity was measured by incubation
of HeLa cell nuclear extract (a source of histone deace-
tylase enzymes) prepared according to Dignam, J. D.;
Lebovitz, R. M.; Roeder, R. G. Nucleic Acids Res. 1983,
11, 1475, with a [3H]acetate-radiolabeled peptide substrate
followed by extraction with a [3H]acetate-radiolabeled
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released product ([3H]acetic acid) with ethyl acetate and
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