Stereodefined and polyunsaturated inhibitors of histone deacetylase based on (2E,4E)-5-arylpenta-2,4-dienoic acid hydroxyamides

Article abstract of DOI:10.1016/j.bmcl.2004.03.012

Syntheses of (2E,4E)-5-arylpenta-2,4-dienoic acid hydroxyamides are described, some of which are potent inhibitors of histone deacetylase, a double bond conferring more than a 10-fold increase in potency compared with the triple bond analogue oxamflatin.

Full text of DOI:10.1016/j.bmcl.2004.03.012

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2477–2481
Stereodefined and polyunsaturated inhibitors of histone deacetylase
based on (2E,4E)-5-arylpenta-2,4-dienoic acid hydroxyamides
Charles M. Marson,a,* Nawal Serradji,a Alphonso S. Rioja,a Sebastien P. Gastaud,a
John P. Alao,b R. Charles Coombesb and David M. Vigushinb
aDepartment of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H OAJ, UK
bDepartment of Cancer Medicine, 6th Floor MRC Cyclotron Building, Imperial College, Hammersmith Hospital Campus,
Du Cane Road, London W12 ONN, UK
Received 4 December 2003; revised 18 February 2004; accepted 2 March 2004
Abstract—Syntheses of (2E,4E)-5-arylpenta-2,4-dienoic acid hydroxyamides are described, some of which are potent inhibitors of
histone deacetylase, a double bond conferring more than a 10-fold increase in potency compared with the triple bond analogue
oxamflatin. Variation of substituents on the aromatic ring has a marked effect on potency, in vitro IC50 values down to 50 nM being
obtained.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
of an equal or larger number of other genes. Non-
histone proteins including transcription factors, nuclear
hormone receptors and a-tubulin are also targets for
acetylation and there is increasing evidence that acetyl-
ation has an important regulatory role in diverse cellular
processes. In the HDAC enzyme pocket, the chain
portion of the natural product trichostatin A can take
the place of an acetylated lysine residue of histone
protein, the potent inhibition of HDAC by trichostatin
A being consistent with the binding of its hydroxamic
acid unit to a zinc atom in the catalytic pocket of the
enzyme, as shown in a crystal structure of a zinc com-
plex of trichostatin A bound to the histone deacetylase-
like protein HDLP.7
In 1964 was described the first isolation of histone
deacetylase from crude nuclear extracts of cells,1 but
only recently has molecular characterisation of isoforms
of the enzyme been achieved.2 Inhibitors of histone
deacetylase (HDAC) enzymes are a new and promising
class of anticancer agents, able to regulate transcription
and inhibit cancer cell proliferation by induction of cell
cycle arrest, differentiation and/or apoptosis.3 Reversible
post-translational acetylation of e-amino groups of
highly conserved lysine residues in nucleosome core
histones is important in the modification of chromatin
topology and gene transcription.4–6 Acetylation status is
controlled by the opposing activities of histone acetyl-
transferase (HAT) and HDAC enzymes. HDAC inhib-
itors induce histone hyperacetylation associated with
transcriptional activation of certain genes but repression
As part of our anticancer programme centred on ther-
apy using small molecules, we have identified novel
antileukaemic agents.8a;b By using structural features
O
O
O
O
OH
N
H
OH
OH
N
H
N
H
O O
S
N
Ph
N
R
Oxamflatin
Trichostatin A
H
* Corresponding author. Tel.: +44(0)20-7679-4712; fax: +44(0)20-7679-7463; e-mail: c.m.marson@ucl.ac.uk
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.012

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C. M. Marson et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2477–2481
present in the natural product trichostatin A, a potent
inhibitor of HDAC, we sought to design and synthesise
novel small-molecule therapeutic agents.9 It was of
interest to ascertain whether HDAC activity could still
be obtained by formal excision of the propionyl unit
present in trichostatin A, and whether improved meta-
bolic stability might also result. We were encouraged by
the moderate HDAC inhibitory activity reported for
oxamflatin, a mammalian antitumour agent that causes
morphological reversion of cancer cells10 and induces
activation of the transcription factor Jun D.11 Oxam-
flatin contains an enyne group linking the aromatic
portion and the hydroxamic acid terminus. Here, we
report that a trans,trans-1,3-butadiene linkage is indeed
compatible with potent HDAC inhibition, a 10-fold
improvement on oxamflatin (IC50 ¼ 2 lM) being readily
achieved, and we describe the synthesis and activities of
some arylpenta-2,4-dienoic acid hydroxyamides with
IC50 values extending down to 50 nM.
(81%). Treatment of esters 3b and 3c in methanol with a
mixture of 50% aqueous hydroxylamine (10 equiv) and
KOH (3.0 equiv) in methanol (addition at 0 °C over
30 min followed by a further period at 0 °C for 30 min,
then stirring for 16 h at 20 °C; procedure A) afforded the
hydroxamic acids 4b (59%) and 4c (66%) after recrys-
tallisation from ethyl acetate.
The parent compound 4a was prepared in 83% yield by
reaction of cinnamaldehyde with (triphenyl-c5-phosph-
anylidene)acetic acid ethyl ester (1.2 equiv) in toluene at
40 °C for 6 days to give the ester 3a, which was con-
verted into hydroxamic acid 4a (45%) using 50% aque-
ous hydroxylamine and KOH as above (procedure A)
but with a final period of 48 h at 20 °C.
A diene hydroxamic acid containing a sulfonamide lin-
ker capable of extending into the ‘cap’ region of histone
deacetylase was secured by the reduction of the nitro
compound 3e to the arylamine 5 (83%) by stirring with a
mixture of FeSO4Æ7H2O (12 equiv), 0.880 aqueous
ammonia (6 mL/mmol arylamine) and ethanol (8 mL/
mmol arylamine) at 60 °C for 10 min (procedure B),
over-reduction being avoided under those conditions.
Reaction of 5 with p-methoxybenzenesulfonyl chloride
(1.5 equiv) in pyridine at reflux for 24 h afforded the
sulfonamide 6 (90%), which was converted into the
hydroxamic acid 4d (62%) using a mixture of 50%
aqueous hydroxylamine and KOH as above (procedure
A, but with a final period of 48 h at 20 °C).
2. Chemistry
Few 5-arylpenta-2,4-diene acid hydroxyamides are
known, and the most general method suffers from very
low yields in the Wittig olefination of aromatic alde-
hydes.12 (2E,4E)-5-Arylpenta-2,4-diene acid hydroxy-
amides that lack further substitution at the 2-, 3-, 4- and
5-positions, and their corresponding carboxylic acid and
ester precursors present challenges since (a) conven-
tional Wittig olefinations are often not efficient, (b) the
diene configuration must be controlled and (c) appro-
priately substituted cinnamaldehdyes may be inaccessi-
ble. We sought to overcome all those problems by
reaction of an aromatic aldehyde with a stabilised
arsenic ylid (Scheme 1), the latter being known for
achieving high (E)-selectivity.13 Reaction of aromatic
aldehydes 1b, 1c and 1e in THF with the ylid derived
Syntheses of (2E,4E)-4-methyl-5-arylpenta-2,4-dienoic
acid hydroxyamides with (E,E)-selectivity were conve-
niently achieved by means of an aldol condensation with
concomitant dehydration, followed by a Wittig olefin-
ation (Scheme 2). The known14 (E)-aldehydes 7b, 7c and
7d were reacted with (triphenyl-c5-phosphanylid-
ene)acetic acid ethyl ester (8) to give the (2E,4E)-dienic
esters 9b,15 9c and 9d in respective yields of 40%, 63%
and 92%. Hydrolysis of 9b with NaOH (2.0 equiv) in
ethanol (portionwise addition of water until turbid, at
intervals over the reflux period of 2 h) gave after neu-
tralisation the corresponding dienic acid (96%), which
was treated with oxalyl chloride (1.1 equiv) and the acid
chloride reacted with 50% aqueous hydroxylamine in
from
3-ethoxycarbonylallylidenetriphenylarsonium
bromide, using 2.0 equiv K2CO3 for 1b (7 days at 0 °C
then 10 min at 20 °C) and 1.1 equiv n-BuLi for 1c and 1e
(20 min at 0 °C then 10 min at 20 °C) afforded the cor-
responding dienic esters 3b (83%), 3c (51%) and 3e
O
O
O
H
2
Ph3As
OH
OEt aq. NH2OH
N
H
OEt
O
R
R
R
1b R = Cl
1c R = NMe2
1e R = NO2
4a R = H 4b R = Cl 4c R = NMe2
3
4d R = NHSO C H p-
OMe
2 6 4
Fe2SO4.7H2O,
0.880 ammonia
50% aq. NH2OH,
KOH
(R = NO2)
SO2Cl
O
O
OEt
OEt
MeO
pyridine,
6
H2N
5
HN
SO2
reflux
MeO
Scheme 1. Synthesis of (2E,4E)-5-arylpenta-2,4-dienoic acid hydroxyamides.

C. M. Marson et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2477–2481
2479
H
OEt
O 8
O
H
H
O
50% aq.
NH2OH,
O
Ph3P
OH
OEt
O
N
H
O
KOH
NaOEt
O
R
R
R
R
1
9b R = Cl
7b R = Cl
10b R = Cl
9d R = NO2
7c R = NMe2
7d R = NO2
10c R = NMe2
O
O
SO2Cl
OH
50% aq.
OEt
N
H
OEt
OH
O O
O O
NH2OH,
S
S
R
N
H
H2N
N
H
12e R = Cl
11
10d R = H
pyridine,
reflux
KOH
10e R = Cl
10f R = OMe
R
R
Fe2SO4.7H2O,
0.880 ammonia
LiOH
(R = Cl)
O
O
OEt
O O
S
N
O2N
13
9d
H
Cl
Scheme 2. Synthesis of (2E,4E)-4-methyl-5-arylpenta-2,4-dienoic acid hydroxyamides.
THF to give the hydroxamic acid 10b (35%). The direct
conversion of ester 7c into hydroxamic acid 10c was
more satisfactory, a 47% yield being achieved using a
mixture of 50% aqueous hydroxylamine (10 equiv) and
KOH (3.0 equiv) in methanol (procedure A).
by a p-chloro group (4b); surprisingly, a p-dimethyl-
amino group as part of the simple aryldiene structure is
much less potent (1.7 lM). Evidently, this p-dimethyl-
amino group does not occupy the same region of the
enzyme as does trichostatin A, presumably because 4b
possesses a shorter chain length. In view of the 4-methyl
group present in trichostatin A, the p-chloro and
p-dimethylamino derivatives 10b and 10c were tested;
while 10c is appreciably more active than 4c the desired
potency was not achieved.
Reduction of the nitro ester 9d by stirring with a mixture
of FeSO4Æ7H2O (12 equiv), 0.880 aqueous ammonia
(6 mL/mmol arylamine) and ethanol (8 mL/mmol aryl-
amine) at 60 °C for 10 min afforded the aromatic amine
11 (92%) that proved to be a versatile intermediate in the
synthesis of a variety of novel sulfonamide-containing
hydroxamic acids 10d–f. Reaction of 11 with an arene-
sulfonyl chloride (2.0 equiv) in pyridine at reflux for 2 h
gave the corresponding sulfonamides 12d (73%), 12e
(44%) and 12f (83%); those were converted into the
respective hydroxamic acids 10d (69%), 10e (44%) and
10f (59%) using a mixture of 50% aqueous hydroxyl-
amine (10 equiv) and KOH (3.0 equiv) in methanol.
Hydrolysis of ester 12e with LiOH (5.0 equiv) in 1:1
THF–water at 70 °C for 16 h gave the acid 13 (91%). All
intermediates and products were isolated in exclusively
the desired (E,E)-configuration.
The moderate potency of the simple arylpentadiene series
was ascribed to a combination of a short chain length and
the lack of a group that could extend into the ‘cap’ region.
At this point, the convenience of the amine intermediates
5 and 11 was exploited by sulfonylation, with the choice
of a relatively lipophilic terminus, in view of the pre-
dominantly hydrophobic region of histone deacetylase
near the periphery of the catalytic ‘tunnel’. The par-
ent sulfonamide 10d showed promising potency
(IC50 ¼ 172 nM);
moreover
the
corresponding
p-chloro and p-methoxy derivatives 10e and 10f showed
excellent potency (IC50 ¼ 49 and 74 nM, respectively).
Comparison of 10f with 4d (IC50 ¼ 10.4 mM) shows that
a 4-methyl group can increase in vitro potency by over
two orders of magnitude, a remarkable observation that
has some parallel with the potency of trichostatin A
(IC50 ¼ 5 nM), which also contains substitution at both
the positions C-4 and C-6. Further investigation of this
observation is in progress. The unsaturated chain com-
pounds 4b and 10e were found to be, respectively, 10 and
24 times more potent than their saturated counterparts 14
and 15. Lastly, the carboxylic acid 13 was far less potent
than 10e, in keeping with the much weaker binding of
zinc to carboxylate compared with hydroxamate.
Catalytic hydrogenation of the esters 3b and 9d in
methanol at 20 °C over 10% Pd–C (up to 2 h) afforded
the corresponding saturated esters (91% and 95%,
respectively), which were converted into the respective
hydroxamic acids 14 (60%) and 15 (54%) using a mix-
ture of 50% aqueous hydroxylamine (10 equiv) and
KOH (3.0 equiv) in methanol.
3. Biological results and discussion
Table 1 shows the IC50 values for the inhibition of
histone deacetylase16 of various compounds described
above. The parent diene hydroxamic acid 4a shows
appreciable potency (IC50 ¼ 279 nM) that is little altered
In conclusion, the efficacy of arylpenta-2,4-dienoic acid
hydroxyamides indicates that neither the keto group nor
the full seven-carbon chain in trichostatin A is essential for

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C. M. Marson et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2477–2481
Table 1. In vitro inhibition of histone deacetylase16
of histone deacetylase herein described are notable for
their nonpeptidic nature, which may confer improved in
vivo stability over the more common amidic inhibitors
that can be cleaved by peptidases. Additionally, 10e was
shown to inhibit proliferation of breast cancer cell lines
at low micromolar concentrations.
Compd Structure
HDAC
IC50 (nM)
O
OH
4a
4b
279 46
252 83
N
H
O
OH
N
H
Acknowledgements
Cl
O
Financial support for the Mandeville Trust and a sti-
pend from L’Association de la Recherche sur le Cancer
(to N.S.) is gratefully acknowledged.
OH
N
4c
1700 200
302 204
H
N
O
OH
10b
N
H
References and notes
Cl
1. Allfrey, V.; Faulkner, R. M.; Mirsky, A. E. Proc. Natl.
Acad. Sci. U.S.A. 1964, 51, 786.
2. For the identification of histone deacetylase HD1, see:
Taunton, J.; Collins, J. L.; Schreiber, S. L. J. Am. Chem.
Soc. 1996, 118, 10412.
O
OH
N
10c
788 334
H
N
3. (a) Johnstone, R. W. Nat. Rev. Drug Discov. 2002, 1, 287;
(b) Johnstone, R. W. Curr. Med. Chem. 2001, 8, 1505; (c)
Jung, M. Curr. Med. Chem. 2001, 8, 1505.
4. Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074.
5. Taunton, J.; Hassig, C. A.; Schreiber, S. L. Science 1996,
272, 408.
O
OH
N
H
14
2580 520
Cl
O
6. Darkin-Rattray, S. J.; Gurnett, A. M.; Myers, R. W.;
Dulski, P. M.; Crumley, T. M.; Allocco, J. J.; Cannova,
C.; Meinke, P. T.; Colletti, S. L.; Bednarek, M. A.; Singh,
S. B.; Goetz, M. A.; Dombromski, A. W.; Polishook, J.
D.; Schmatz, D. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
13143.
7. Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.;
Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P.
Nature 1999, 401, 188.
8. (a) Rioja, A.; Pizzey, A. R.; Marson, C. M.; Thomas, N. S.
B. FEBS Lett. 2000, 467, 291; (b) Marson, C. M.;
Vigushin, D. M.; Rioja, A.; Coombes, R. C. Bioorg.
Med. Chem. Lett. 2002, 12, 255.
9. Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.;
Breslow, R.; Rifkind, R. A.; Marks, P. A. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 3003.
10. Kim, Y. B.; Lee, K.-H.; Sugita, K.; Yoshida, M.;
Horinouchi, S. Oncogene 1999, 18, 2461.
11. Sonoda, H.; Nishida, K.; Yoshioka, T.; Ohtani, M.;
Sugita, K. Oncogene 1996, 13, 143.
12. Ohtani, M.; Matsuura, K.; Shirahase, K.; Sugita, K.
J. Med. Chem. 1996, 39, 2871.
13. Huang, Y.; Shen, Y.; Zheng, J.; Zhang, S. Synthesis 1985,
57.
OH
N
H
O O
S
10d
172 38
N
H
O
OH
N
H
O O
S
N
10e
10f
49 4
H
Cl
O
O
OH
OH
N
O O
S
74 16
H
N
H
MeO
MeO
N
H
O O
S
4d
15
10,400 1300
1170 50
N
H
O
O
OH
N
H
O O
S
N
H
Cl
Cl
14. The following procedures were used. For 7b: Goodwin, T.
E.; Ratcliff, D. G.; Crowder, C. M.; Seitzinger, N. K.
J. Org. Chem. 1982, 47, 815; For 7c: Sunjic, V.; Majeric,
M.; Hamersak, Z. Croat. Chem. Acta 1996, 69, 643; For
7d: Hirata, H.; Nakata, H.; Yamada, K.; Okuhara, K.;
Naito, T. Tetrahedron 1961, 14, 252.
OH
O O
S
13
>100,000
N
H
15. Cappon, J. J.; Boart, J.; Walle, G. A. M.; Lugtenburg, J.
Recl. Trav. Chim. Pays-Bas 1991, 5, 158.
16. Vigushin, D. M.; Ali, S.; Pace, P. E.; Mirsaidi, N.; Ito, K.;
Adcock, I.; Coombes, R. C. Clin. Cancer Res. 2001, 7, 971;
Histone deacetylase activity was measured by incubation of
HeLa cell nuclear extract (a source of histone deacetylase
potent enzyme inhibition to be achieved. Also confirmed
is that trans,trans-stereochemical rigidity is desirable,
presumably providing a locking of conformation similar
to that conferred by the trans,trans-configuration and
zig-zag backbone of trichostatin A. The novel inhibitors

C. M. Marson et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2477–2481
2481
enzymes) prepared according to: Dignam, J. D.; Lebovitz,
R. M.; Roeder, R. G. Nucl. Acids Res. 1983, 11, 1475, with
a [3H]acetate-radiolabelled peptide substrate followed by
extraction of the released product ([3H]acetic acid) with
ethyl acetate and quantification by liquid scintillation
counting. The substrate was a synthetic peptide corre-
sponding to the N-terminal residues 14–21 of histone H4
that had been chemically acetylated in vitro with [3H]acetic
acid sodium salt (3.7 GBq/mmol; New England Nuclear
Boston, MA) as described in Ref. 5 above. The concentra-
tion of compound that inhibited histone deacetylase
enzymatic activity by 50% (IC50) was determined graphi-
cally in each case using nonlinear regression analysis to fit
inhibition data to the appropriate dose–response curve
(GraphPad Prism Version 3.0; GraphPad Software Inc.
San Diego, CA). Each test compound was assayed in
duplicate whilst positive control (trichostatin A) and
negative control samples were assayed in triplicate.