Aromatic sulfide inhibitors of histone deacetylase based on arylsulfinyl-2,4-hexadienoic acid hydroxyamides

Article abstract of DOI:10.1021/jm051010j

The synthesis of a novel series of potent inhibitors of histone deacetylases is described, based on arylsulfinyl-2,4-hexadienoic acid hydroxyamicles and their derivatives. In vitro IC₅₀ values clown to 40 nM were obtained, and several compounds showed inhibition of CEM (human leukemic) cell viability with IC₅₀ of ～1.5 μM, comparable to or better than that of suberoylatiilide hydroxamic acid, an inhibitor of histone cleacetylase currently in clinical trials.

Full text of DOI:10.1021/jm051010j

800
J. Med. Chem. 2006, 49, 800-805
Aromatic Sulfide Inhibitors of Histone Deacetylase Based on Arylsulfinyl-2,4-hexadienoic Acid
Hydroxyamides
Charles M. Marson,*,† Pascal Savy,† Alphonso S. Rioja,† Thevaki Mahadevan,† Catherine Mikol,‡ Arthi Veerupillai,‡
Eva Nsubuga,‡ Angela Chahwan,‡ and Simon P. Joel‡
Department of Chemistry, UniVersity College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H OAJ, U.K., and
Department of Medical Oncology, St. Bartholomew’s Hospital, London W12 ONN, U.K.
ReceiVed October 8, 2005
The synthesis of a novel series of potent inhibitors of histone deacetylases is described, based on arylsulfinyl-
2,4-hexadienoic acid hydroxyamides and their derivatives. In vitro IC50 values down to 40 nM were obtained,
and several compounds showed inhibition of CEM (human leukemic) cell viability with IC50 of ∼1.5 µM,
comparable to or better than that of suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase
currently in clinical trials.
Introduction
have entered clinical trials. The efficacy of such HDAC
inhibitors is generally considered to depend on the presence of
(1) a terminal group (hydroxamic acid in the present class of
novel inhibitors) that can bind to the metal-containing active
site of HDAC enzymes and is connected to (2) a chain of several
atoms (“linker”) that is linked to (3) an end moiety (or “cap”)
of sufficient bulk to occupy the cleft outside the pocket that
leads to the active site. Such a model is in agreement with crystal
structures of histone deacetylase-like protein (HDLP) to which
is bound TSA or SAHA.20
As part of our anticancer program centered on therapy using
small molecules, we have used the framework of trichostatin
A to design novel HDAC inhibitors (II).21 We have shown that
HDAC inhibitory potency can be maintained (in vitro IC50 down
to 50 nM)21 by using a simplified carbon chain although it lacks
the keto group and adjacent chiral center present in trichostatin
A. As HDAC mimics of trichostatin A, a number of function-
alities have been interposed between an aromatic ring and an
alkyl or alkylene chain, especially numerous amides,20,22-25 and
recently an oxygen atom in the form of the aromatic ether
designated A-161906, which showed potent in vitro HDAC
inhibition (IC50 ) 9 nM) but appeared to have correspondingly
less satisfactory in vivo properties.26 HDAC inhibitors based
on aromatic thioethers were not reported and have the advantage
of an adjustable oxidation level at sulfur for improving activity,
possibly by means of a prodrug mode of action. Such replace-
ment of carbon by sulfur was also structurally appealing since
a sulfur isostere, the sulfoxide of 12, could be prepared and
evaluated in which a sulfoxide group replaced the inherently
reactive carbonyl group of trichostatin A. Accordingly, we
describe here the synthesis and preliminary biological evaluation
of a new class of sulfur-containing HDAC inhibitors, principally
of type I.
Inhibitors of histone deacetylase (HDAC) enzymes are
emerging as a promising class of anticancer agents1,2 able to
regulate gene transcription3-5 and inhibit cancer cell proliferation
by induction of cell cycle arrest, differentiation, and/or
apoptosis.6-8 HDAC enzymes catalyze the deacetylation of
ꢀ-amino groups of lysine residues in the N-terminal tails of core
histones in the nucleosome, resulting in an increased interaction
of the protonated ꢀ-amino lysine residues with DNA, leading
to a compacted and less accessible chromatin structure.9
Consequently, HDACs can act as repressors of gene transcrip-
tion by mediating conformational changes in the nucleosome
and altering the accessibility of transcription factors to DNA.10,11
The inappropriate recruitment of HDAC enzymes by oncogenic
proteins may alter gene expression in favor of arrested dif-
ferentiation and/or excessive proliferation.12 Accordingly, the
hyperacetylation of chromatin induced by inhibitors of HDACs
usually results in the relief of transcriptional repression of certain
genes, e.g., the cyclin-dependent kinase inhibitor protein
p21WAF1/CIP1 12,13
Relief of transcriptional repression is known
.
to be central to the therapeutic mode of action of HDAC
inhibitors in the treatment of several proliferative diseases,
including a variety of leukemias.14,15
Chemistry
Few arylsulfanylhexa-2,4-dienoic acids are known,27 and to
our knowledge no arylsulfanylhexa-2,4-dienoic acid hydroxya-
mides have been previously reported. Reaction of methyl sorbate
and N-bromosuccinimide in chlorobenzene at reflux and under
irradiation with a sunlamp afforded methyl (2E,4E)-6-bromo-
2,4-hexadienoate (2).28 Ester 2 or the corresponding ethyl ester
proved suitable for alkylation of a variety of aromatic thiols,
typically in tetrahydrofuran and in the presence of triethylamine
(1.0 equiv) at 20 °C or where the reaction was more sluggish
A number of small-molecule inhibitors of HDAC including
trichostatin A (TSA)16 and suberoylanilide hydroxamic acid
(SAHA)17 induce differentiation in cancer cell lines and suppress
cell proliferation. Several HDAC inhibitors including SAHA,
NVP-LAQ824,5 MS-275,18 and the cyclodepsipeptide FK-22819
* To whom correspondence should be addressed. Phone: +44(0)20 7679
4712. Fax: +44(0)20 7679 7463. E-mail: c.m.marson@ucl.ac.uk.
† University College London.
‡ St. Bartholomew’s Hospital.
10.1021/jm051010j CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/21/2005

Inhibitors of Histone Deacetylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 801
Scheme 1a
a Reagents: (a) Et3N, Bu4NI, THF; (b) aqueous NH2OH; (c) NaIO4. Key: (a) R ) H (n ) 1); (b) R ) Cl (n ) 1); (c) R ) OMe (n ) 1); (d) R ) NMe2
(n ) 1); (e) R ) H (n ) 2); (f) Ar ) 2-naphthyl.
Scheme 2a
Scheme 3a
a Reagents: (a) NaOMe, MeOH, THF; (b) H2, Pd/C; (c) 37% aqueous
HCHO, NaBH3CN, ZnCl2, MeOH; (d) LiAlH4, THF; (e) pyridine-SO3,
Et3N, DMSO; (f) Ph3PdCHCO2Et, CH2Cl2; (g) 50% aqueous NH2OH, 1
M KOH in MeOH, THF.
a Reagents: (a) Et3N, Bu4NI, THF; (b) DIBAL, THF; (c) pyridine-
SO3, Et3N, DMSO; (d) Ph3PdCHCO2Et, CH2Cl2; (e) 50% aqueous NH2OH,
1 M KOH in MeOH, THF.
Scheme 4a
by heating at reflux (Scheme 1). The resulting esters 3 were
readily converted into the corresponding sulfoxides 5 (n ) 1)
by treatment with NaIO4 (1.2 equiv) in aqueous methanol; the
sulfone 5e was prepared by reaction of methyl (2E,4E)-6-bromo-
2,4-hexadienoate (2) with sodium benzenesulfinate. The thio-
ethers 3 and the sulfoxides 5 (n ) 1) were converted into their
corresponding hydroxamic acids 4 and 6 by treatment of a
solution of the appropriate ester in THF with 50% aqueous
hydroxylamine (9 equiv) at 0 °C to which was subsequently
added a solution of potassium hydroxide in methanol (1 M, 1.6
equiv) with stirring over ∼30 min. The corresponding carboxylic
acids of the thioethers 3 and the sulfoxides 5 (n ) 1) could be
prepared by hydrolysis with lithium hydroxide or sodium
hydroxide.
a Reagents: (a) NaH, DMF; (b) H2, Pd/C, EtOAc, then 37% aqueous
HCHO, NaBH3CN, ZnCl2, MeO; (c) LiAlH4, THF; (d) pyridine-SO3, Et3N,
DMSO; (e) (EtO)2P(O)CH2CHdCHCO2Et, NaH, DME; (f) 50% aqueous
NH2OH, 1 M KOH in MeOH, THF.
Synthesis of the trichostatin thio analogue 12 (Scheme 2)
required a sequence compatible with the sensitive nitrogen,
sulfur, and dienic functionality. 4-Dimethylaminobenzenethiol
(1d) was reacted with methyl (E)-4-bromo-2-methyl-2-pen-
tenoate (7) in the presence of triethylamine to give the key
thioether 8 in 95% yield. Ester 7 was prepared by reaction of
methyl (E)-2-methylpent-2-enoate with N-bromosuccinimide in
carbon tetrachloride at reflux and under irradiation with a
sunlamp.31 Reduction of ester 8 by diisobutylaluminum hydride
(3 equiv) at 0 °C in toluene afforded the corresponding allylic
alcohol 9 (88%), which was subjected to Moffatt oxidation
(pyridine-sulfur trioxide complex in DMSO followed by
addition of triethylamine) to give a solution from which the
aldehyde 10 was extracted with diethyl ether; evaporation
afforded the crude aldehyde 10, which was reacted with ethyl
(triphenylphosphoranylidene)acetate (1.1 equiv) in THF to give
the ethyl ester 11 in 76% yield after purification by column
chromatography. Treatment of ester 11 with 50% aqueous
hydroxylamine (9 equiv) followed by a solution of potassium
hydroxide in methanol gave the trichostatin thio analogue 12
in 52% yield.
substituted hydroxamic acid 12, was of considerable interest.
The likely difficulty in forming an aryl ether linkage by reaction
of thiol 1d with an arene precursor that would be relatively
unactivated suggested the reverse combination, i.e., a thiol
component such as 14 for reaction with a displaceable halogen
atom, as in p-chloronitrobenzene (13). Such an approach would
necessitate conversion of the nitro group into the dimethylamino
group present in diarylthioether 19. The sequence in Scheme 3
proved to be effective; thus, treatment of the thiolate of ester
14 in methanol with p-chloronitrobenzene (13) first at 0 °C
followed by heating at reflux for 2 h gave the thioether 15
(73%), which was reduced quantitatively to the corresponding
amino compound, which was reductively aminated with aqueous
37% formaldehyde in the presence of NaBH3CN and ZnCl2 to
give the key N,N-dimethylamino intermediate 16 in 80% yield.
The latter was then reduced quantitatively with LiAlH4 to give
the alcohol 17, which was subjected to a sequence of Moffat
oxidation, Wittig olefination, and hydroxamation, similar to that
described above for alcohol 9 and resulting in the novel
cinnamohydroxamic acid 19.
Since the cinnamohydroxamic acid moiety was shown to be
a favorable linker and terminus for HDAC inhibitors, the
diarylthioether 19, a benzannulated version of the 1,3-dimethyl-
In the quest for a more druglike compound (Scheme 4), a
fused system, benzo[b]thiophene, was considered in which a
methyl group in the chain was formally annealed to the aromatic

802 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Marson et al.
Scheme 5a
Table 1. In Vitro Inhibition of Histone Deacetylase and CEM Cell
Viability
a Reagents: (a) Et3N; (b) 50% aqueous NH2OH, 1 M KOH in MeOH,
THF; (c) NaIO4. Key: (a) R1 ) H, R2 ) Me, n ) 1; (b) R1 ) Cl, R2 )
Me, n ) 1; (c) R1 ) Cl, R2 ) Et, n ) 2; (d) R1 ) NMe2, R2 ) Me, n )
1; (e) R1 ) p-ClC6H4SO2NH, R2 ) Me, n ) 1; (f) R1 ) NH2, R2 ) Me,
n ) 1.
ring present in the previous compound types. The ready
accessibility of the suitably functionalized benzo[b]thiophene
22,32 together with previously successful sequences of chain
extension and hydroxamation permitted Scheme 4 to be realized.
Thus, ester 22, prepared from sodium hydride, ethyl 2-mercap-
toacetate, and 2-chloro-5-nitrobenzaldehyde, was hydrogenated
over 10% palladium on carbon to give the corresponding amine
that after filtration was used directly in solution for the reductive
amination with aqueous 37% formaldehyde in the presence of
NaBH3CN and ZnCl2 to give the key N,N-dimethylamino
intermediate 23 in 60% yield. The latter was reduced with
LiAlH4 to give the alcohol 24, which was subjected to a
sequence of Moffat oxidation, Wittig olefination (differing from
the above sequences by the use of triethyl phosphonocrotonate),
and hydroxamation, similar to that described above for alcohols
9 and 1 and resulting in the novel cinnamohydroxamic acid 27.
A variety of compounds containing a saturated chain (Scheme
5) were prepared using methods similar to those for the
unsaturated ones shown in Scheme 1. Thus, thiolates 1 were
reacted with ω-bromoesters 28 to give thioethers 29 that could
be converted into the corresponding sulfoxides 31 using NaIO4.
Treatment of 29 and 31 with 50% aqueous hydroxylamine and
potassium hydroxide, as described above, afforded the corre-
sponding hydroxamic acids 30 and 32. Treatment of sulfide 30b
with NaIO4 afforded the sulfoxide 32b in 68% yield. Upon
treatment with m-CPBA, sulfoxide 32a also underwent oxida-
tion, affording the corresponding sulfone 33 in 54% yield. Such
oxidations at sulfur in the presence of a hydroxamic acid group
provide a useful alternative to establishing the desired oxidation
level at sulfur prior to conversion into the hydroxamic acid.
Biology
Table 1 shows that most of the novel sulfur-containing
compounds exhibited HDAC IC50 of <400 nM in the purified
enzyme assay,33 a promising result since the assay generally
gives appreciably higher IC50 than assays involving release of
tritiated acetic acid from an N-acylated peptide substrate.4,5 The
HPLC-based HDAC assay was modified to permit the deter-
mination of HDAC activity in intact cells at a single time-point,
reflecting uptake of the compound by cells and inhibition of
intracellular HDAC enzymes. Moreover, this measure of cellular
HDAC inhibitory activity showed a better correlation with the
concentration of inhibitor that induced a 50% reduction in cell
viability in CEM cells after a 72 h incubation than with values
from the liver HDAC assay (Spearman’s rank correlation
coefficient for liver IC50 vs % viability IC50 ) -0.603, p )
0.017; cell IC50 vs % viability IC50 correlation coefficient )
a ND ) not determined.
0.82, p < 0.001). The cellular HDAC assay may therefore be
a more accurate indication of the desired biological endpoint
for further screening of HDAC inhibitors that show activity
against partially purified enzyme.
The low IC50 value for CEM cell viability (1.6 µM) for 4b
encouraged a further study using a modification of the whole-
cell assay to allow the determination of intracellular HDAC
inhibitory activity after 1 and 24 h of incubation in CEM cells
in culture media. The concentration of 4b required to maintain
50% HDAC inhibition after 24 h was not different from the

Inhibitors of Histone Deacetylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 803
Summary
concentration at 1 h (3.4 vs 3.4 µM). Additionally, similar
studies with 32b showed comparable stability to 4b (2.9 µM at
1 h vs 3.4 µM at 24 h). This is in contrast to SAHA, which
required a 65% increase in the concentration to achieve 50%
HDAC inhibition at 24 h (0.9 vs 2.6 µM), and in marked contrast
to TSA for which a >90% increase in concentration was
required (0.03 vs 0.37 µM). Those data suggest an improved
metabolic stability of 4b in CEM cells.
The efficacy of arylpenta-2,4-dienoic acid hydroxyamides
indicates for the first time that aromatic thioether units can be
present in a novel class of potent inhibitors of histone deacety-
lase. The sulfides tested, although structurally less similar to
trichostatin A than the corresponding sulfones, were generally
more potent than the latter, the sulfides probably exhibiting
greater cellular uptake than the more polar sulfoxides (or
sulfones). Sulfide 4b was appreciably more metabolically stable
than SAHA and far more so than trichostatin A. Since the rat
liver assay22 generally gives significantly higher in vitro IC50
than do assays based on deacetylation of tritiated histone-like
substrates,4,5 the present values of under 200 nM for several
compounds are considered to be promising. Additionally, 32b
was shown to inhibit proliferation of three cell lines by 50% at
0.9-2.7 µM. One disadvantage of amidic HDAC inhibitors that
have been clinically tested is their potential for cleavage by
endogenous peptidases, a feature that is absent in most of the
novel inhibitors of histone deacetylase herein described and that
may confer improved in vivo stability over the more common
amidic inhibitors such as SAHA.
All S-compounds in Table 1 except 32c contain five carbon
atoms between the sulfur atom and the carbon atom of the
hydroxamic acid moiety, the additional methylene group in 32c
(compared with 32b) conferring lower activity in all three assays
and indicating that a linker of six methylene groups may be
longer than optimal; that would be consistent with the additional
length of ∼0.8 Å arising from two carbon-sulfur bonds,
compared with the two carbon-oxygen bonds present in
A-161906. Both dienic and fully saturated chains were found
to possess potent in vivo HDAC inhibitory activities. Potencies
of the parent systems (entries 1-3) are high in the enzyme assay
but low in both CEM cell assays, a pattern also found for the
unsubstituted and unsaturated counterparts (entries 4 and 5).
Of the para substituents examined, chloro was usually very
effective (entries 7, 11, and 14) in the CEM cell assays.
However, additional methyl groups as in 12 did not improve
HDAC inhibitory activity, hydroxamic acid 12 showing an IC50
of 0.34 µM in the HDAC liver assay and 5.3 µM in CEM cells.
Despite the sulfur atom of (()-12 being a replacement of the
carbonyl group in trichostatin A, the latter (as a single
enantiomer) has a much greater potency in the HDAC liver assay
(IC50 ) 0.015 µM) and the CEM cell asays (IC50 ) 0.019 µM
for HDAC inhibition and 0.12 µM for % viablility). In the
compounds studied, the presence of a p-N,N-dimethylamino
group was generally consistent with moderate but not outstand-
ing potencies (entries 8, 13, and 17).
Experimental Section
Chemistry. Melting points were determined on a microscope
hot-stage apparatus and are uncorrected. IR spectra were recorded
1
on a Perkin-Elmer PE-983 spectrophotometer. H and 13C NMR
spectra were recorded on a Bruker AC300 instrument at 300 and
75 MHz, respectively; chemical shifts are reported in δ (ppm)
relative to the internal reference tetramethylsilane. The homogeneity
of the compounds was monitored by ascending thin-layer chroma-
tography, performed on Merck 0.2 mm aluminum-backed silica gel
60 F254 plates and visualized using an alkaline KMnO4 spray or by
ultraviolet light. Flash column chromatography was performed using
Merck 0.040-0.063 mm, 230-400 mesh silica gel. Elemental
analysis (C, H, N, Cl, and S) results were obtained on a Perkin-
Elmer 2400 CHN elemental analyzer, and all data were consistent
with theoretical values (within (0.4%) unless otherwise indicated.
Mass spectra were obtained on a VG7070H mass spectrometer using
Finigan Incos II. All solvents were reagent grade and, where
necessary, were purified and dried by standard methods. Evapora-
tion refers to the removal of solvent under reduced pressure. Flash
column chromatography was performed using Sorbsil C60 40/60A
silica gel. The following compounds were prepared according to
the literature: (2E,4E)-6-phenylsulfanyl-2,4-hexadienoic acid,27a
methyl (2E,4E)-6-phenylsulfanyl-2,4-hexadienoate,39 methyl (2E,4E)-
6-bromo-2,4-hexadienoate (2),28 (2E,4E)-6-benzenesulfinyl-2,4-
hexadienoic acid,30 (2E,4E)-6-benzenesulfonyl-2,4-hexadienoic acid,30
(2E,4E)-6-benzenesulfonyl-2,4-hexadienoic acid hydroxyamide,30
4-dimethylaminobenzenethiol (1d),29,30 (E)-methyl 2-methyl-2-
pentenoate,35 with methyl 2(E)-4-bromo-2-methyl-2-pentenoate
(7),31 5-nitrobenzo[b]thiophene-2-carboxylic acid ethyl ester (22),32
methyl (2E,4E)-2,4-hexadienoate,34 3-mercaptobenzoic acid,36 meth-
yl 3-mercaptobenzoate (14),37 methyl 6-bromohexanoate (28a),38
ethyl 6-phenylsulfanyl hexanoate (29a),39 N-(4methyl-7-coumari-
nyl)-N-ω-(tert-butyloxycarbonyl)-N-ω-acetyllysinamide (MAL),33
(5-acetylamino-1-carbamoylpentyl)carbamic acid tert-butyl ester.30
(2E,4E)-6-(4-Chlorophenylsulfanyl)-2,4-hexadienoic Acid Hy-
droxyamide (4b). Procedure A. To a solution of methyl (2E,4E)-
6-(4-chlorophenylsulfanyl)-2,4-hexadienoate (0.44 g, 1.64 mmol)
in distilled THF (9.0 mL) containing 50% aqueous hydroxylamine
(1.0 mL, 15.2 mmol) was added at 0 °C a solution of potassium
hydroxide in methanol (1 M, 2.6 mL, 2.6 mmol) over 30 min. After
the mixture was stirred at 0 °C for 1 h, distilled water (9.0 mL)
was added and the mixture was made neutral by dropwise addition
of 2 M HCl at 0 °C. The aqueous solution was extracted with ethyl
acetate (3 × 30 mL), and the combined extracts were dried over
anhydrous MgSO4 and evaporated. The residue was recrystallized
from acetone to give 4b (0.21 g, 48%) as pale-brown microprisms,
Although the sulfoxides were generally more potent than the
corresponding sulfides in the enzyme assay (entries 1, 2; 4, 5;
6, 7 but contrast 11, 14), sulfide 4b and sulfoxide 6b were
of comparable potency in the CEM cell HDAC assay. The
sulfide 4f, containing a hydrophobic capping region, is also of
notable potency. The effect of sulfoxide 32b on a further three
cell lines was investigated; 32b was shown to be cytotoxic to
HCT116 (percentage viability IC50 ( SD: 3.9 ( 0.7 µM), SUD4
(7.5 ( 1.1 µM), and MCF-7 (6.1 ( 0.9 µM) cells. The
concentration of 32b that inhibited proliferation (cell number)
by 50% in those cells was 0.9-2.7 µM. The saturated chain
sulfides 30b and 30e showed potent activities using the purified
enzyme assay but performed very poorly in the CEM cell assays;
in contrast, the saturated chain sulfoxides 32b-d typically
showed good activities in all three assays. The striking difference
in CEM cell activity between 30b and 32b may indicate the
importance of a polar entity adjacent to the aromatic ring (such
as the keto group in TSA) or may be associated with a more
rapid cellular metabolism of the saturated chain sulfides. The
additional hydrophobicity provided by the chlorophenyl terminus
in 30e was found, as expected,21 to confer improved in vitro
enzyme inhibition. While the cinnamohydroxamic acid type 19
provides tolerably good activities, such was not the case for 27
in which a fused ring system is present. Overall, these studies
suggest that a sulfide or a sulfoxide linkage as part of an alkyl
or possibly an alkylene chain, together with an extended
hydrophobic capping region, could provide good potency and
good antiproliferative properties with comparable or better
metabolic stability than SAHA and much greater metabolic
stability than TSA.

804 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Marson et al.
mp 120-122 °C (dec). 1H NMR (CDCl3, 400 MHz) δ 10.63 (br s,
1H), 8.95 (br s, 1H), 7.34 (s, 4H), 6.98 (dd, J ) 15.0, 11.2 Hz,
1H), 6.29 (dd, J ) 15.0, 11.2 Hz, 1H), 6.07 (m, 1H), 5.75 (d, J )
15.2 Hz, 1H), 3.75 (d, J ) 7.1 Hz, 2H); 13C NMR (CDCl3, 100
MHz) δ 162.7, 137.8, 135.1, 134.4, 131.1, 130.7, 130.4, 128.9,
122.0, 34.5. Anal. (C12H12NClO2S) Calcd: C 53.43%, H 4.48%,
N 5.19%. Found: C 52.88%, H 4.59%, N 5.08%.
the pH was adjusted to 6-7 by dropwise addition of aqueous 1 M
NaOH. The organic layer was separated and washed with saturated
aqueous ammonium chloride (10 mL) and then with brine (10 mL).
The organic layer was dried over anhydrous MgSO4, filtered, and
evaporated. The oily residue was recrystallized from acetone to
give 34 (0.18 g, 54%) as white microprisms, mp 184-186 °C. 1H
NMR (300 MHz, DMSO-d6) δ 10.19 (br s, 1H, OH), 8.68 (br s,
1H, NH), 7.70-7.55 (m, 5H), 2.95 (m, 1H), 2.87 (m, 1H), 1.90 (t,
J ) 7.1 Hz, 2H), 1.70-1.20 (m, 6H); 13C NMR (75 MHz, DMSO-
d6) δ 168.8, 143.2, 135.3, 129.2, 125.9, 55.1, 31.9, 27.4, 24.6, 21.0.
Anal. (C12H17NO4S) C, H, N.
(2E,4E)-6-(Naphthalen-2-ylsulfanyl)-2,4-hexadienoic Acid Hy-
droxyamide (4f). To a solution of methyl (2E,4E)-6-(naphthalen-
2-ylsulfanyl)-2,4-hexadienoate (0.505 g, 1.78 mmol) in distilled
THF (10 mL) containing 50% aqueous hydroxylamine (1.08 mL,
16.4 mmol) was added at 0 °C a solution of potassium hydroxide
in methanol (1 M, 2.5 mL, 2.5 mmol) over 30 min. Continuation
as in procedure A gave a residue that was recrystallized from ethyl
acetate to give 4f (0.315 g, 62%) as white microprisms, mp 141-
Biology. HDAC Enzyme Assay. The procedure described in
Hoffmann33 was modified for use as described below and was
further modified for use in the whole-cell HDAC activity assay.
Isolated Liver HDAC Activity Assay. The supernatant resulting
from the high-speed centrifugation of rat liver homogenates was
partially purified on a Q-Sepharose column using a sodium chloride
gradient (10-500 mM), with a Tris (15 mM), EDTA (0.25 mM),
glycerol (10%) buffer at pH 7.9. Fractions were collected and
analyzed for HDAC activity using the method described herein.
Those fractions containing HDAC activity were pooled and aliquots
stored at -40 °C prior to use. The assay mixture comprised 100
µL of pooled HDAC enzyme, 100 µL of buffer (Tris-HCl (10 mM),
NaCl (10 mM), MgCl2 (15 mM), EGTA (0.1 mM), glycerol (10%
v/v), and mercaptoethanol (0.007%), 100 µL of HDAC inhibitor
(dissolved in DMSO and diluted with HEPES buffer (50 mM, pH
7.4) to working concentrations), and 100 µL of substrate (MAL,
diluted to 20 µg/mL with 50 mM HEPES buffer, pH 7.4, 0.5 mL
aliquots stored at -40 °C, diluted to 5 µg/mL for use in the assay).
Samples were vortexed for 15 s and incubated at 37 °C for 60 min
with gentle agitation every 15 min. Acetonitrile (100 µL) was then
added to terminate the reaction, after which samples were centri-
fuged at 10 000 rpm for 10 min and then placed on ice prior to
analysis by HPLC of substrate (MAL) and deacetylated product
(ML) concentration in the supernatant.
Whole-Cell HDAC Activity Assay. The 1 × 106 CEM cells in
RPMI medium (1 mL; Sigma Chemicals, Poole, Dorset U.K.) were
exposed to HDAC inhibitors at six concentrations for 60 min at 37
°C, after which MAL (20 µg/mL final concentration) was added
and the mixture incubated for a further 30 min. Cells were then
rapidly washed with phosphate buffered saline at 4 °C and lysed
by sonication. The reaction was stopped with acetonitrile (100 µL),
and intracellular MAL and ML concentrations in the supernatant
were determined by HPLC analysis.
1
142 °C. H NMR (CDCl3, 300 MHz) δ 7.82-7.43 (m, 7H), 7.37
(m, 1H), 6.28 (m, 2H), 5.97 (d, J ) 15.4 Hz, 1H), 3.74 (d, J ) 5.9
Hz, 2H); 13C NMR (CDCl3, 75 MHz) δ 167.5, 145.3, 142.8, 143.9,
139.3, 135.8, 133.8, 133.4, 132.6, 131.1, 129.5, 121.6, 51.9, 37.4.
Anal. (C16H15NO2S) C, H, N.
(2E,4E)-6-(4-Chlorobenzenesulfinyl)-2,4-hexadienoic Acid Hy-
droxyamide (6b). To a solution of methyl (2E,4E)-6-(4-chloroben-
zenesulfinyl)-2,4-hexadienoate (0.80 g, 2.79 mmol) in distilled THF
(15 mL) containing an aqueous solution of hydroxylamine (50%,
1.7 mL, 25.8 mmol) was added at 0 °C a solution of potassium
hydroxide in methanol (1 M, 4.5 mL, 4.5 mmol) over 30 min. After
the mixture was stirred at 0 °C for 1 h, distilled water (15 mL)
was added and the mixture was made neutral by dropwise addition
of 2 M HCl at 0 °C. Workup as for procedure A gave a residue
that was recrystallized from acetone to give 6b (0.45 g, 56%) as
pale-brown microprisms, mp 159 °C (dec). 1H NMR (CDCl3, 300
MHz) δ 0.68 (br s, 1H), 9.21 (br s, 1H), 7.62 (s, 4H), 6.97 (dd, J
) 15.0, 11.2 Hz, 1H), 6.23 (dd, J ) 15.0, 11.2 Hz, 1H), 5.81 (d,
J ) 15.0 Hz, 1H), 5.79 (m, 1H), 3.88 (dd, J ) 13.0, 7.7 Hz, 6H),
3.65 (dd, J ) 13.0, 8.0 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ
162.5, 142.1, 137.4, 136.0, 135.6, 129.1, 127.1, 126.2, 123.0, 58.6.
Anal. (C12H12NClO3S) C, H, N, S.
6-Phenylsulfanylhexanoic Acid Hydroxyamide (30a). To a
stirred solution of ethyl 6-phenylsulfanylhexanoate (0.40 g, 1.59
mmol) at 0 °C in anhydrous THF was added a solution of
hydroxylamine (1 M, 16 mL) in methanol, followed immediately
by dropwise addition of a solution of potassium hydroxide (1 M,
2.07 mL) in methanol. The mixture was warmed to 25 °C and stirred
for 16 h. Ice was then added and the pH adjusted to 6-7 by addition
of 2 M HCl. Workup as in procedure A gave a residue that was
recrystallized from acetone to give 30a (0.26 g, 68%) as white
microprisms, mp 132-135 °C. 1H NMR (300 MHz, DMSO-d6) δ
10.03 (br s, 1H), 8.78 (br s, 1H), 7.41-7.30 (m, 5H), 2.90 (t, J )
7.1 Hz, 2H), 1.88 (t, J ) 7.1 Hz, 2H), 1.50-1.32 (m, 4H), 1.27
(m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 168.8, 136.1, 130.0,
129.3, 129.0, 32.3, 32.0, 28.7, 27.7, 24.7. Anal. (C12H17NO2S) C,
H, N.
Quantitation of MAL and ML by HPLC Analysis. Chromato-
graphic separation of MAL and ML was carried out using a 15 cm
Apex ODS 5 µm column with an acetonitrile/distilled water (40:
60), 2% TFA (v/v) mobile phase at a flow rate of 1.2 mL/min.
MAL and ML were quantified by fluorescence detection at
excitation/emission wavelengths of 330/395 nm. The activity of
each inhibitor was assessed at a minimum of five nonzero
concentrations, determined from initial concentration studies. The
peak heights of MAL and ML were used to derive the % MAL in
the mixture as the ratio MAL/(MAL+ML). The % MAL in the
absence of inhibitor (typically 22-25%) was taken as 100% HDAC
activity, and the % HDAC activity at each inhibitor concentration
6-(Benzenesulfinyl)hexanoic Acid Hydroxyamide (32a). To a
stirred solution of ethyl 6-(benzenesulfinyl)hexanoate (0.50 g, 1.87
mmol) at 0 °C was added a solution of hydroxylamine (1 M, 18.7
mL) in methanol, followed immediately by dropwise addition of a
solution of potassium hydroxide (1 M, 2.43 mL) in methanol. The
mixture was warmed to 25 °C and stirred for 16 h. Ice was then
added and the pH adjusted to 6-7 by addition of 2 M HCl. Workup
as in procedure A gave a residue that was recrystallized from
acetone to give 32a (0.31 g, 65%) as white microprisms, mp 164-
was derived from (100 - % MALDRUG)/(100 - % MALNODRUG
)
× 100. These data (minimum of n ) 4 at each concentration for
each inhibitor) were fitted to a sigmoidal EMAX model (Graphpad
Prism, version 2.01) to derive the IC50 concentration for each
compound.
1
165 °C. H NMR (300 MHz, DMSO-d6) δ 10.09 (br s, 1H), 8.71
(br s, 1H), 7.60 (m, 5H), 3.02 (t, J ) 7.2 Hz, 2H), 2.20 (t, J ) 7.2
Hz, 2H), 1.47-1.35 (m, 4H), 1.28 (m, 2H); 13C NMR (75 MHz,
DMSO-d6) δ 168.9, 136.4, 130.1, 129.8, 128.5, 54.1, 32.2, 29.4,
27.7, 25.0. Anal. (C12H17NO3S) C, H, N.
6-(Benzenesulfonyl)hexanoic Acid Hydroxyamide (33). To a
stirred solution of 6-(benzenesulfinyl)hexanoic acid hydroxyamide
(0.31 g, 1.22 mmol) in anhydrous dichloromethane (6.0 mL) at 0
°C was added m-CPBA (0.21 g, 1.22 mmol) in small portions. The
mixture was maintained at 0 °C for 1 h with stirring, after which
Cytotoxicity Assay of HDAC Inhibitors. CEM (human leu-
kemic) cells were exposed to HDAC inhibitors at a minimum of
five nonzero concentrations for 3 days under standard cell culture
conditions (RPMI medium, 37 °C, 5% CO2 in a humidified
atmosphere). Cell number and percentage viability were assessed
by trypan blue staining. The % viability IC50 was determined using
Graphpad Prism. For 32b these studies were extended to include
HCT116 (colorectal cancer), MCF-7 (breast cancer), and SUD4
(lymphoma) cells.

Inhibitors of Histone Deacetylase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 805
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Acknowledgment. Financial support from the Lewis Family
Charitable Trust, St. Bartholomew’s and the Royal London
Charitable Foundation, and the Nikki Taylor Memorial Fund is
gratefully acknowledged.
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Supporting Information Available: Experimental procedures,
1H and 13C NMR spectra for all new compounds, and microana-
lytical data. This material is available free of charge via the Internet
at http://pubs.acs.org.
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