Polymer-assisted, multi-step solution phase synthesis and biological screening of histone deacetylase inhibitors

Article abstract of DOI:10.1039/b313414h

The polymer-assisted solution phase synthesis (PASP) of an array of histone deacetylase (HDAc) inhibitors is described. HDAc inhibitors have considerable potential as new anti-proliferative agents. Selected compounds were shown to inhibit both human endothelial cell proliferation, and the formation of tubules (neovascularisation) in an in vitro model of angiogenesis.

Full text of DOI:10.1039/b313414h

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Polymer-assisted, multi-step solution phase synthesis and biological
screening of histone deacetylase inhibitors
Akanksha Bapna,a Emma Vickerstaffe,b Brian H. Warrington,b Mark Ladlow,*b
Tai-Ping D. Fan*a and Steven V. Ley*c
a Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge,
UK CB2 1QJ
b GlaxoSmithKline Cambridge Technology Centre, University Chemical Laboratory,
Lensfield Road, Cambridge, UK CB2 1EW. E-mail: Mark.2.Ladlow@gsk.com;
Fax: ϩ44 (0)1223 331532; Tel: ϩ44 (0)1223 336349
c Department of Chemistry, University Chemical Laboratory, Lensfield Road, Cambridge,
UK CB2 1EW
Received 24th October 2003, Accepted 16th December 2003
First published as an Advance Article on the web 27th January 2004
The polymer-assisted solution phase synthesis (PASP) of an array of histone deacetylase (HDAc) inhibitors is
described. HDAc inhibitors have considerable potential as new anti-proliferative agents. Selected compounds were
shown to inhibit both human endothelial cell proliferation, and the formation of tubules (neovascularisation) in an
in vitro model of angiogenesis.
Introduction
In recent years, the parallel synthesis of focused arrays of
analogues of ‘lead’ structures has evolved into a powerful
method to facilitate the rapid determination of structure activ-
ity relationships (SAR) and thus drive the lead optimisation
process in the pharmaceutical industry.1 To achieve this object-
ive, attention has increasingly concentrated on the adoption of
efficient and versatile solution-phase synthesis approaches, as
opposed to solid-phase organic synthesis (SPOS)2 strategies. In
this way, difficulties associated with SPOS, such as unaccept-
ably long reaction development and optimisation times, the
need to use large reagent excesses to drive reactions to comple-
tion, and the inability to perform the purification of resin-
bound intermediates, are largely circumvented. A particularly
powerful and widely adopted approach to parallel solution
of cell differentiation and proliferation. However, an imbalance
phase synthesis utilises polymer-supported reagents and scaven-
ger resins.3 Polymer-assisted solution phase (PASP) synthesis
reduces reaction work-ups to a series of simple filtrations and
facilitates in-line purification by solid-phase extraction and
‘catch and release’ techniques.4 It is thus well-suited to enable
the rapid, multi-step synthesis of focused compound arrays.
Recently, we have reported a fully automated, multi-step
synthesis using polymer supported reagents to prepare a series
of histone deactylase (HDAc) inhibitors5 based upon the
reported lead structure 1a.6 These are structurally related to the
natural product trichostatin A (TSA) 27 and suberoylanilide
hydroxamic acid (SAHA) 3.8 Here we describe in detail the
development of the polymer-assisted synthesis, and the manual
preparation and associated biological activity of a 24-member
array of HDAc inhibitors 1a–x.
There is increasing evidence that the acetylation and deacetyl-
ation of proteins and small molecules is an important regu-
latory modification in many cellular processes.9 In particular,
the reversible acetylation of the ε-amino functionalities of con-
served lysine residues in the N-terminal tails of nucleosomal
histones results in chromatin remodelling and is a key biological
event controlling gene transcription.10 This post-translational
modification of histones is co-regulated by a combination of
histone acetyltransferase (HAT) and histone deacetylase
(HDAc) enzymes, that cause conformational changes in the
nucleosome, altering the accessibility and binding of transcrip-
tion factors to DNA. In normal cells, this leads to a regulation
in the level of histone acetylation has been associated with
malignant disease.11 HDAc inhibitors lead to a reversal of
transcriptional repression and an associated upregulation of
tumour suppressors.12
In addition, HDAc inhibitors have been observed to result in
an inhibition of angiogenesis13 and are therefore of consider-
able interest as potential new anti-cancer agents.11c Other
studies have suggested that HDAc inhibitors may also offer
promise as novel anti-protozoal agents14 and new anti-virals.15
Results and discussion
Chemistry
Previous work has established that the hydroxamic acid func-
tionality in TSA coordinates to a zinc cation that is held by
several hydrogen bonds in the active site of HDLP (histone-
deacetylase-like-protein), a bacterial homologue of eukaryotic
HDAcs.16 The polyene chain in TSA acts as a spacer group that
occupies a lipophilic channel leading from the zinc binding
domain of the enzyme to a region occupied by the aromatic
capping group. Therefore, we elected to prepare a focused array
based upon 1a that retained the core aryl propenoic hydroxamic
acid functionality but incorporated three points of diversity in
order to investigate the consequences of: (i) sulfonamide
N-methylation, (ii) regioisomerism about the aniline, and
(iii) different substituents on the terminal benzenesulfonamide.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 1 1 – 6 2 0
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Scheme 1 Reagents and conditions: a) (i). DMF, 50 ЊC, 1 h, ×2; (ii). SCX SPE purification; b) MeI, DMF, rt, 18 h; c) acrylic acid, triethylamine,
DMF, 90 ЊC, 18 h; d) (i). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, DMF, rt, 20 min; (ii). hydroxyl-
amine-OTHP, 50 ЊC, 18 h; (iii). NH2 SPE purification; e) Amberlyst H-15, MeOH, rt, 2 h.
Moreover, we wished to develop a multi-step synthesis that did
not require aqueous work-ups and could be realised using only
a sequence of immobilised reagents and scavenger resins. In
addition, we recognised that the ability to use the same solvent
for all transformations would further simplify reaction work-up
and establish a route that would subsequently be amenable to
full automation.
Initially, we developed a suitable polymer-assisted procedure
for each step of the synthesis in turn before combining
these protocols to prepare a sample of 1a in a flow-through
manner, without isolation or chromatographic purification of
any intermediates (Scheme 1). Finally, the array 1a–x was
assembled in a similar way by parallel polymer-assisted
multi-step synthesis.
Considering the reported synthesis of 1a, we wished to
avoid the need to first prepare a bis-sulfonylated product which
then required hydrolysis back to the desired mono-sulfonamide.
However, although we have reported the sulfonylation of amines
using sulfonyl chlorides immobilised on polymer-supported
4-dimethylaminopyridine (PS-DMAP),17 the sulfonylation of
anilines in this way was not precedented. In the event, treatment
of the 4-iodoaniline 4 with the supported reagent 5, prepared
from toluenesulfonyl chloride and PS-DMAP at reflux in di-
chloromethane, gave the desired toluenesulfonamide 6 in 35%
isolated yield. No evidence of bis-sulfonamide formation was
observed, but the presence of the toluenesulfonic acid derived
from hydrolysis of the immobilised reagent was evident. A simi-
lar result was obtained using acetonitrile or tetrahydrofuran
as solvent. However, the use of dimethylformamide proved
optimal giving rise to no observable toluenesulfonic acid by-
product and a moderate increase in yield to 40%. Ultimately, we
determined that repeating this protocol twice using a fresh
batch of pre-formed PS-DMAP/tosyl chloride followed by
filtration through an Isolute SCX cartridge18 to scavenge un-
reacted aniline, gave the desired sulfonamide 6 in 64% yield and
excellent purity (>95% by HPLC and 1H-NMR).
encapsulated palladium acetate (Pd–EnCat)20 and a variety
of fibrous polystyrene-supported palladium phosphine com-
plexes (FibreCat)21 were profiled in combination with a limit-
ing amount of acrylic acid using a ReactArray SK233 auto-
mated reaction sampling system.22 In general, we observed that
reducing the amount of Pd catalyst present led, unsurprisingly,
to slower conversions, but more significantly, an increase in the
amount of the corresponding reduced product 7 derived from
the competing reduction pathway was also observed. Under
the conditions examined, the use of Pd–EnCat resulted in the
highest conversion to 9 with least contamination by 7. In the
event, 7 could be readily removed from the crude reaction
product by a single recrystallisation. Alternatively, the crude
product could be progressed directly to the next stage of the
synthesis because 7 does not participate in the subsequent
hydroxmate formation and may be removed at a later stage.
Initially, formation of the hydroxamate 11 in high
yield proved problematic. A number of coupling reagents
including 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU), 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDC), and diisopropylcarbo-
diimide (DIC) were examined under a variety of conditions
for their ability to effect this condensation. Ultimately, optimal
conditions were determined to be pre-activation in the presence
of EDC for 20 min, followed by the introduction of hydroxyl-
amine tetrahydropyranyl ether and subsequent incubation at
50 ЊC. After non-aqueous work-up by filtration through an
amino-SPE cartridge, 11 was obtained in a respectable 70%
isolated yield and excellent purity (90% by LC-MS). Finally,
the THP O-protecting group was removed by treatment with
Amberlyst H-15 in the presence of added methanol to give the
desired hydroxamic acid 1a in good purity and 71% isolated
yield.
In a similar way, Heck olefination of the N-Me analogue 8
with acrylic acid gave the acid 10 (80% yield). This material was
converted into the corresponding intermediate tetrahydro-
pyranyl hydroxamate 12 which was deprotected directly to
afford the N-Me hydroxamic acid 1b in 76% yield and 95%
LC-MS purity.
Having established that the complete synthesis could be
performed in DMF without the need for aqueous work-up
or the evaporation of solvents at intermediate stages, we next
attempted the synthesis of 1a in a flow-through manner. In a
linear synthesis, the need to efficiently wash resins after each
step without subsequent solvent evaporation tends to lead to a
Next, we turned our attention to the N-methylation of the
sulfonamide in order to introduce a second point of diversity.
This transformation could be reliably achieved with iodo-
methane in dimethylformamide in the presence of the polymer-
supported phosphazene base PS-BEMP19 to cleanly afford 8 in
90% isolated yield.
In order to facilitate product clean-up in the subse-
quent Heck olefination of 6 with acrylic acid, we elected to
use an immobilised source of palladium. A polyurea micro-
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Table 1 Monomer sets chosen for combinatorial array 1a–x
it was noted that in the Heck olefination step, tert-sulfonamides
tended to lead to lower purity products than secondary sulfon-
amides. Nevertheless, all the desired products were obtained
in reasonable overall yields with acceptable purities (Fig. 2).
However, to obtain reliable SAR it is desirable to assay sub-
strates with uniformly high purities. Therefore, prior to screen-
ing, a single auto-preparative purification step was performed
to ensure that all substrates had purities in excess of 95%.
R1-SO2Cl:
R2:
H
Me
I-R3-NH2:
dilution of the reaction solution which, in turn, can have a
detrimental effect on reaction rates and yields. Therefore, we
initiated the flow-through synthesis at a higher substrate
concentration than used previously and carefully minimised
resin wash solvent volumes. Using the validated reaction
conditions, the complete synthesis was performed in a flow-
through manner without any intermediate solvent removal or
off-line purification of intermediates to afford the hydroxamic
acid 1a in 47% overall isolated yield and 87% purity according
Fig. 2 Isolated yields and purities for crude array compounds 1a–x.
Biology
The in vitro biological activity of the compounds 1a–x was
measured at 1 µM in a primary HDAc enzyme inhibitory assay
using HeLa cell nuclear extract (principally HDAc-1 and 2).
The readout was obtained as the amount of deacetylated
substrate produced and is inversely proportional to enzyme
inhibitory activity. Since HDAc inhibitors are known to induce
cell death in cell types that cannot maintain a G1/G2 check-
point, the ability of the compounds to inhibit HUVEC (human
umbilical vein endothelial cell) proliferation was also deter-
mined.23 In this assay, the number of viable cells remaining after
incubation with the inhibitor for 48 h was measured spectro-
photometrically following introduction of the MTS reagent.24
Endothelial cells play a central role in angiogenesis, which is
essential for the growth of solid tumours beyond a few cubic
millimeters and hence, the most active analogues were also
evaluated for their ability to inhibit tubule formation in a
HUVEC-fibroblast co-culture which is an in vitro model of
angiogenesis. In this model, the endothelial cells initially form
small islands within the culture matrix. The HUVECs subse-
quently begin to proliferate and then enter a migratory phase
during which they move through the matrix to form tubules.
These tubules eventually join over a period of 12–14 days to
form a network of anastomosing tubes that closely resembles a
capillary bed (Fig. 3). The significance of angiogenesis in the
growth of tumours and the distribution and progression of
metastatic disease makes vascular targeting an important
strategy for the effective, long term control of cancers.25
1
to LC-MS and H NMR analysis. The purity profile for this
reaction sequence is shown in Fig. 1.
Results and discussion
In vitro, the most effective enzyme inhibitors (i.e. those com-
pounds producing deacetylated substrate in the range 10–20
µM) were found to be the toluenesulfonamide 1a, the bi-
phenylsulfonamide 1k and the 3,4-dimethoxysulfonamide 1q
(Table 2). Notably, in this assay, the 3,4-dimethoxy analogue 1q
was found to be equipotent with TSA. Whilst compounds
1a and 1q are derived from a para-substituted R3 core motif,
compound 1k, derived from a meta-substituted core, was more
potent than its para-analogue 1i. The weakest compounds were
the thiophenyl analogues 1u, 1w and 1x.
Fig. 1 Purity profile for the flow-through synthesis of hydroxamic acid
1a.
With a reliable synthetic route in hand, we next selected a
series of monomers to prepare a small focused compound array
1a–x in order to investigate SAR associated with this pharma-
cophore (Table 1). Initially, a set of six polystyrene-supported
sulfonyl pyridinium chlorides were prepared. For a small com-
pound array such as 1a–x, it was feasible to perform reaction
quality control by LC-MS throughout the synthesis. In this way,
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1a and 1b, were administered to HUVECs at higher concen-
trations (>20 µM), a decrease in the number of cells below that
present at the start of the assay was observed implying that
cell death was also occurring at these concentrations.26 These
observations suggest that the overall anti-proliferative action
of HDAc inhibitors such as 1a and 1c in HUVECs is a com-
bination of a specific inhibition of cell proliferation and an
induction of cell death.
Compounds 1a, 1c and TSA were also assayed in vitro for
their ability to inhibit tubule formation in a HUVEC-fibroblast
co-culture (Angiokit).27 All of these compounds were found to
be effective in reducing total vessel area. Compounds 1a and 1c
were essentially equipotent and effective at sub-micromolar
concentrations, whereas TSA was approximately 10 fold more
potent and was an effective inhibitor below 10 nM (Fig. 3).
Interestingly, both TSA and 1a are effective at inhibiting angio-
genesis at <100 nM, a concentration below that at which a
significant anti-proliferative effect was observed in isolated
HUVECs.
Conclusions
In summary, we have prepared an array of hydroxamic acids
1a–x which are structurally related to TSA, using a parallel,
multi-step polymer-assisted solution phase (PASP) synthesis.
Following biological evaluation in vitro, a number of these
compounds were found to possess micromolar inhibitory activ-
ity in a HeLa cell nuclear extract enzyme inhibition assay
(HDAc-1 and 2), and anti-proliferative activity in human
umbilical vein endothelial cells (HUVECs). In addition, the
most active compounds 1a and 1c were shown to be potent
inhibitors of tube-formation (neovascularisation) in an in vitro
model of angiogenesis.
Fig. 3 TSA and 1a inhibit tube-formation in an in vitro angiogenesis
assay. The HUVEC-fibroblast co-culture was treated with increasing
concentrations of TSA and 1a and immunocytochemically stained with
PECAM-1 antibodies after 11 days. The images were analysed using
Angiosys software and the total tubule area was measured in bit map
units. A. Blank; B. Negative control (2 mM suramin); C, E TSA; D, F
Compound 1a; G, H Graph of mean tubule area. Values are SEM
measured from 8 microscope fields for each concn.
This study serves to further demonstrate the utility of PASP
synthesis techniques for the rapid preparation of focused,
biologically relevant compound arrays.
Experimental
Synthesis
In general, N-methylation of the sulfonamide nitrogen led to
a reduction in enzyme inhibitory activity. For example, com-
pounds 1o, 1s and 1q were more effective inhibitors than their
N-Me counterparts 1p, 1t and 1r. An exception to this observ-
ation arises when contrasting the analogues 1i (N-H) and 1j
(N-Me) where 1j is more potent than 1i.
All solvents and reagents were used as supplied unless other-
wise stated. Where appropriate, reactions were carried out
under an inert atmosphere of nitrogen. Analytical thin layer
chromatography (TLC) was performed on Merck silica gel 60
F254 pre-coated plates. Analytical high pressure liquid chrom-
atography (HPLC) was performed using a Hewlett Packard
Series 1050 instrument. Column: Supelcosil ABZϩPLUS 3.3
cm × 4.6 mm, 3 µm. Eluent A: water, 0.1% TFA, B: acetonitrile
95%, water 5%, TFA 0.05%. Flow rate: 1 cm3 minϪ1. Detection:
UV (diode array: 215, 230, 254 nm). Method: gradient 10–95%
B in A over 7 min. Preparative HPLC was performed using a
Gilson auto-preparative instrument. Column: Supelcosil
ABZϩPLUS 10.0 cm × 2.12 cm, 5 µm. Eluent A: water, 0.1%
TFA, B: acetonitrile 95%, water 5%, TFA 0.05%. Flow rate: 6 ml
minϪ1. Detection: UV (254 nm). Method: gradient 10–95% B in
A over 25 min. Infrared spectra were collected on a Perkin-
Elmer Spectrum One FT-IR instrument under attenuated total
reflectance (ATR) conditions. Liquid chromatography-mass
spectra (LC-MS) were recorded using a Hewlett Packard Series
1050 HPLC attached to a Micromass ZQ 2000 mass spectro-
meter under electrospray positive and negative ionisation con-
ditions. Column: Supelcosil ABZϩPLUS 3.3 cm × 4.6 mm,
3 µm. Eluent A: 10 mM solution of ammonium acetate in water,
0.1% formic acid, B: acetonitrile 95%, water 5%, formic acid
0.05%. Flow rate: 3 ml minϪ1. Detection: UV (diode array: 215–
330 nm). Method: gradient 0–100% B in A over 3.5 min. High
resolution mass spectra were obtained using a Micromass
Q-TOF 2 under positive or negative ionisation conditions.
NMR spectra were recorded on Bruker AM 400 or Bruker
Considering the MTS in vitro cell proliferation assay, a num-
ber of compounds were found to possess micromolar anti-
proliferative activity. However, whilst the thiophenyl analogue
1w, for example, was one of the weakest enzyme inhibitors in
HeLa nuclear extract, it was determined to be a potent anti-
proliferative agent in HUVECs. The most potent anti-prolifer-
ative agents in vitro were the toluenesulfonamides 1a (para-R3
core) and 1c (meta-R3 core) both with IC50s of 2 µM. The corre-
sponding 4-biphenyl analogues 1k (meta-core) and 1i (para-
core) were also effective with IC50s of 4 and 3 µM respectively.
Interestingly, in the case of 1i, the corresponding N-methylated
analogue 1j was also a potent anti-proliferative agent in
HUVECs.
The lack of correlation between the in vitro enzyme inhib-
ition and anti-proliferative assays suggests that in HUVECs the
primary mode of action of 1a–x is likely to be inhibition of
HDAcs other than HDAcs-1 and 2. In addition, in whole cell
assay barriers to biological activity may be presented by the
need to effectively penetrate cellular and/or nuclear membranes
to access the site of action. Therefore, whilst there is no doubt
that compounds such as 1a and 1c are potent anti-proliferative
agents in HUVECs, the precise mode of biological action can-
not be ascribed with certainty at this stage and is the subject of
ongoing studies. However, it was noted that when compounds
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Table 2 Comparison of enzyme inhibitory and anti-proliferative activity of HDAc inhibitors 1a–x
HeLa
HUVEC
IC50
HeLa
HUVEC
extracts
extracts
IC50
Compound R1
R3
R2
H
(µM)a
(µM)b
Compound
R1
R3
R2
H
(µM)a
(µM)b
1a
19
33
32
45
54
34
50
56
58
31
20
35
2
6
1m
29
ND
38
57
13
43
34
54
72
40
61
75
>20
15
1b
1c
1d
1e
1f
1g
1h
1i
Me
H
1n
1o
1p
1q
1r
Me
H
2
>20
13
Me
H
10
Me
H
>20
>20
>20
>20
3
8
Me
H
Me
H
29
1s
>20
17
Me
H
1t
Me
H
1u
1v
1w
1x
>20
ND
3
1j
Me
H
4
Me
H
1k
1l
4
Me
>20
Me
>20
a Amount of deacetylated Color de Lys substrate produced after 30 min. b Inhibition of HUVEC proliferation determined by spectrophotometric
measurement of viable cell count after 48 h following introduction of MTS reagent.
DRX 500 spectrometers at 400 MHz and 500 MHz respectively
was heated at 50 ЊC for 1 h. The reaction was filtered and the
resin again washed with DMF (0.5 ml × 2). The combined
filtrates were applied to an SCX cartridge (0.5 g) and eluted
with methanol to give the sulfonamide 6 as a white solid (24 mg,
64%). Mp: 171–172 ЊC; Rf 0.35 [EtOAc/hexane (1 : 2)]; Found:
C, 41.93; H, 3.07; N, 3.79; S, 8.55%. C13H12NO2IS requires C,
41.84; H, 3.24; N 3.75; S, 8.59%; HRMS: C13H12NO2IS requires
(MH)ϩ 373.9712, found 373.9717; IR νmax/cmϪ1: 3220 (NH),
1
for H NMR, and at 100 MHz and 125 MHz for 13C NMR
(proton decoupled) in the indicated solvent. Chemical shifts
(δ) are reported in parts per million (ppm). The following
abbreviations are used for multiplicities: s = singlet; d = doublet;
t = triplet; m = multiplet; dd = doublet of doublets; br = broad;
and coupling constant J-values are quoted in Hz. Melting
points were measured on a Mettler FP5 automatic melting
point apparatus in open capillaries and are uncorrected. Paral-
lel reactions requiring heating were performed with gentle stir-
ring using a Greenhouse parallel reaction block.28 All other
reactions were performed in fritted plastic filter tubes on a
laboratory shaker.29 Amino and SCX solid-phase extraction
(SPE) cartridges were obtained from Varian.18
1
1320 (SO2), 1160 (SO2); H NMR (400 MHz, CDCl3): δ 10.3
(br s, 1H), 7.61 (d, 2H, J = 8 Hz), 7.52 (d, 2H, J = 8 Hz), 7.31
(d, 2H, J = 8 Hz), 6.86 (d, 2H, J = 8 Hz), 2.30 (s, 3H); 13C
NMR (100 MHz, CDCl3): δ 142.2, 136.2, 134.3, 133.7, 127.7,
125.2, 121.1, 87.1, 19.5; LC-MS (ESI Ϫve): m/z 372 (MH)Ϫ,
tR = 3.58 min; HPLC: tR = 4.79 min (97%; 254 nm).
Polystyrene-supported 4-dimethylaminopyridine-toluene sul-
fonyl chloride adduct (5). Toluenesulfonyl chloride (190 mg, 1.00
mmol) and dried polymer-supported 4-dimethylaminopyridine
(4-DMAP) (0.33 g, 1.00 mmol) were suspended in anhydrous
CH2Cl2 (5 ml). The suspension was agitated for 1 h, filtered,
washed with CH2Cl2 (3 × 5 ml) and then dried in vacuo to give
the polymer-supported reagent 5 as an orange solid (0.47 g).
The combined washings were evaporated and the resin load-
ing was calculated based on the weight of sulfonyl chloride
recovered (2.2 mmol gϪ1).
N-Methyl-N-(4-iodo-phenyl)-4-methyl-benzenesulfonamide
(8). To a solution N-(4-iodo-phenyl)-4-methyl-benzenesulfon-
amide 6 (20.0 mg, 0.05 mmol) in DMF (0.5 ml) was added PS–
BEMP (26 mg, 0.05 mmol) and iodomethane (3.7 µl, 0.06
mmol). The suspension was agitated for 18 h, The resin was
drained, washed with DMF (0.5 ml × 3), and the washings
combined and evaporated in vacuo to give the sulfonamide 8 as
a white solid (17.5 mg, 90%). Mp: 91–92 ЊC; Rf 0.43 [EtOAc/
hexane (1 : 4)]; Found: C, 43.51; H, 3.56; N, 3.58; S, 8.34%.
C14H14NO2IS requires C, 43.42; H, 3.64; N 3.62; S, 8.28%;
HRMS: C14H15NO2IS requires (MH)ϩ 387.9868, found
387.9880; IR νmax/cmϪ1: 1343 (SO2), 1163 (SO2); 1H NMR (400
MHz, CD3OD): δ 7.63 (d, 2H, J = 8 Hz), 7.40 (d, 2H, J = 8 Hz),
7.32 (d, 2H, J = 8 Hz), 6.88 (d, 2H, J = 8 Hz), 3.11 (s, 3H), 2.40
(s, 3H); 13C NMR (100 MHz, CD3OD): δ 146.0, 143.3 139.5,
134.9, 131.1, 129.9, 129.4, 93.2, 38.7, 21.9; LC-MS (ESI ϩve):
m/z 388 (MH)ϩ, tR = 3.78 min; HPLC: tR = 6.23 min (100%; 254
nm).
N-(4-Iodo-phenyl)-4-methyl-benzenesulfonamide (6). To the
PS-DMAP:sulfonyl chloride adduct 5 (45 mg, 0.10 mmol) was
added a solution of 4-iodoaniline (22 mg, 0.10 mmol) in DMF
(1.0 ml). The reaction mixture was heated with gentle stirring at
50 ЊC for 1 h, then filtered and the resin washed with DMF
(0.5 ml × 2). To the filtrate was added an additional quantity of
sulfonyl chloride resin 5 (45 mg, 0.10 mmol) and this mixture
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General conditions for ReactArray study. To the sulfon-
amide 6 (40 mg, 0.10 mmol) and the immobilised palladium
catalyst was added a solution of triethylamine in DMF
(0.66 M × 0.5 ml) and then a solution of acrylic acid in DMF
(0.03 M × 0.5 ml) using a ReactArray SK233 automated work-
station. The reaction mixture was then heated at 90 ЊC with
stirring for a total of 13 h. Aliquots (25 µl) were withdrawn
automatically over this period, diluted with MeCN (0.5 ml) and
subjected to in-line HPLC analysis. Results were plotted as %
composition versus time.
1.8 (m, 3H), 1.5 (m, 3H); LC-MS: (ESI ϩve) m/z 417 (MH)ϩ, tR
= 4.77 min; HPLC: tR = 4.58 min (90%; 254 nm).
N-Hydroxy-3-[4-(toluene-4-sulfonylamino)-phenyl]acrylamide
(1a). Amberlyst H-15 resin (80 mg) was added to a solution of
the THP ether 11 (48 mg, 115 µM) in methanol (3 ml) and the
suspension was agitated at room temperature for 2 h. The resin
was then drained, washed with methanol (0.5 ml × 2) and the
combined filtrates were evaporated in vacuo to afford the
hydroxamic acid 1a as an off-white solid (38 mg, 71%). Mp:
167–168 ЊC; HRMS: C16H16N2O4S (MH)ϩ requires 333.0909,
found 333.0907; IR νmax/cmϪ1: 1649 (C᎐O), 1560 (C᎐C), 1328
3-[4-(Toluene-4-sulfonylamino)-phenyl]-acrylic acid (9). To a
solution of N-(4-iodo-phenyl)-4-methyl-benzenesulfonamide 6
(0.40 g, 1.10 mmol) in DMF (5 ml) was added acrylic acid
(75 µl, 1.10 mmol), triethylamine (0.45 ml, 3.21 mmol) and Pd
EnCat (84 mg, 3 mol% Pd). The reaction mixture was stirred
at 90 ЊC for 18 h, then filtered, and the solvent evaporated
in vacuo to leave a brown gum. Recrystallisation from hexane/
EtOAc afforded the acrylic acid 9 as white crystals (0.29 g,
83%). Mp: 233–234 ЊC; Rf = 0.21 [MeOH/CHCl3 (1 : 19)];
Found: C, 60.53; H, 4.76; N, 4.55; S, 9.94%; C16H15NO4S
requires C, 60.55; H, 4.76; N, 4.41; S, 10.10%; HRMS:
C16H15NO4S requires (MH)ϩ 318.0800, found 318.0809; IR
νmax/cmϪ1: 3255 (NH/OH), 1674 (C᎐O), 1627 (C᎐C), 1322
᎐
᎐
1
(SO2), 1155 (SO2); H NMR (400 MHz, DMSO-d6): δ 10.80
(br s 1H), 10.60 (br s, 1H), 8.93 (br s, 1H), 7.64 (d, 2H,
J = 8 Hz), 7.39 (d, 2H, J = 9 Hz), 7.32 (d, 2H, J = 8 Hz), 7.29 (d,
1H, J = 16 Hz), 7.08 (d, 2H, J = 9 Hz), 6.27 (d, 2H, J = 16 Hz),
2.30 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.3, 145.6,
141.2, 141.1, 138.5, 132.6, 131.1, 130.1, 128.6, 121.9, 117.9,
21.8; LC-MS: (ESI Ϫve) m/z 331 (MH)Ϫ, tR = 2.69 min; HPLC:
tR = 3.92 min (88%; 254 nm).
N-Hydroxy-3-{4-[methyl-(toluene-4-sulfonyl)-amino]-phenyl}-
acrylamide (1b). To a solution of 3-{4-[Methyl-(toluene-4-
sulfonyl)-amino]-phenyl}-acrylic acid 10 (70 mg, 0.21 mmol) in
DMF (1.5 ml), was added a solution of diethylaminopropyl-
carbodiimide hydrochloride (EDC) (52 mg, 0.27 mmol) and
1-hydroxybenzotriazole (HOBt) (42 mg, 0.31 mmol) in DMF
(1.5 ml). The reaction mixture was stirred at room temperature
for 20 min when a solution of o-(tetrahydro-2H-pyran-2-yl)-
hydroxylamine (37 mg, 0.31 mmol) in DMF (500 µl) was added.
The resulting mixture was heated at 50 ЊC for 24 h. Upon
cooling to room temperature, the reaction mixture was applied
to a NH2-SPE cartridge (2 g) and eluted with methanol. To the
resulting methanolic solution of the tetrahydropyranyl ether 12
was added Amberlyst H-15 (300 mg) and this suspension was
shaken at room temperature for 3 h. The suspension was filtered
and the resin was washed with MeOH (2 ml × 2) to afford
the hydroxamic acid 1b as a colourless gum (55 mg, 76%). IR
νmax/cmϪ1: 1656 (C᎐O), 1622 (C᎐O), 1600 (C᎐C), 1345 (SO ),
᎐
᎐
(SO2), 1155 (SO2); 1H NMR (400 MHz, CD3OD): δ 7.66 (d, 2H,
J = 8 Hz), 7.53 (d, 1H, J = 16 Hz), 7.43 (d, 2H, J = 8 Hz), 7.27
(d, 2H, J = 8 Hz), 7.11 (d, 2H, J = 8 Hz), 6.33 (d, 2H, J = 16 Hz),
2.33 (s, 3H); 13C NMR (125 MHz, CD3OD) δ 170.5, 145.4,
145.3, 141.2, 138.1, 131.7, 130.7, 130.2, 128.3, 121.3, 118.4,
21.4; LC-MS: (ESI Ϫve) m/z 316 (MH)Ϫ, tR = 3.04 min; HPLC:
tR = 4.18 min (93%; 254 nm).
3-{4-[Methyl-(toluene-4-sulfonyl)-amino]-phenyl}-acrylic acid
(10). To a solution of N-methyl-(4-iodo-phenyl)-4-methyl-
benzenesulfonamide 8 (85 mg, 0.22 mmol) in DMF (2 ml) was
added acrylic acid (15 µl, 0.21 mmol), triethylamine (91 µl, 0.66
mmol) and Pd EnCat (16 mg, 3 mol% Pd). The reaction mix-
ture was stirred at 90 ЊC for 18 h, then filtered, and the solvent
evaporated in vacuo to leave a brown solid. Recrystallisation
from hexane/EtOAc afforded the acrylic acid 9 as off-white
crystals (54 mg, 80%). Mp: 215–216 ЊC; Rf = 0.21 [MeOH/
CHCl3 (1 : 19)]; HRMS: C17H17NO4S requires (MH)ϩ 332.0957,
found 332.0951; IR νmax/cmϪ1: 1676 (C᎐O), 1625 (C᎐C), 1346
᎐
᎐
᎐
2
1152 (SO2); HRMS C17H18N2O4S requires (MH)ϩ 347.1066,
found 347.1079; 1H NMR (500 MHz, CD3OD): δ 7.58–7.45 (m,
3H), 7.40 (d, 2H, J = 8 Hz), 7.31 (d, 2H, J = 8 Hz), 7.12 (d, 2H,
J = 8 Hz), 6.44 (d, 1H, J = 16 Hz), 3.15 (s, 3H), 2.40 (s, 3H); 13
C
᎐
᎐
(SO2), 1172 (SO2); 1H NMR (400 MHz, CD3OD): δ 7.62 (d, 1H,
J = 16 Hz), 7.39 (d, 2H, J = 8 Hz), 7.40 (d, 2H, J = 8 Hz), 7.32
(d, 2H, J = 8 Hz), 7.15 (d, 2H, J = 8 Hz), 6.44 (d, 1H, J = 16 Hz),
3.16 (s, 3H), 2.40 (s, 3H); 13C NMR (125 MHz, CD3OD)
δ 170.2, 145.6, 145.0, 144.6, 134.6 (2C), 130.6, 129.6, 128.9,
127.6, 120.1, 38.3, 21.5; LC-MS: (ESI ϩve) m/z 332 (MH)ϩ, tR =
3.19 min; HPLC: tR= 5.04 min (97%; 254 nm).
NMR (125 MHz, CD3OD): δ 164.6, 144.1, 142.7, 134.0, 133.6,
133.2, 129.2, 127.7, 127.5, 126.2, 117.7, 36.9, 20.0; LC-MS: (ESI
Ϫve) m/z 345 (MH)Ϫ, tR = 2.83 min; HPLC: tR = 4.21 min (95%;
254 nm).
‘Flow-through’ preparation of N-hydroxy-3-[4-(toluene-4-sul-
fonylamino)-phenyl]acrylamide (1a). To the PS-DMAP:sulfonyl
chloride adduct 5 (65 mg, 0.13 mmol) was added a solution of
4-iodoaniline (29 mg, 0.13 mmol) in DMF (2 ml) and the reac-
tion mixture gently stirred at 50 ЊC for 1 h. The mixture was
cooled, filtered and the resin was washed with DMF (0.5 ml ×
2). The combined filtrates were then added to an additional
quantity of resin 5 (65 mg, 0.13 mmol) and stirred at 50 ЊC for a
further 1 h. Upon cooling to rt, the suspension was drained and
the resin was washed with DMF (0.5 ml). The combined fil-
trates were applied to a SCX cartridge (1 g) which was eluted
with DMF (1.5 ml). The eluate was collected and triethylamine
(35 µl, 0.35 mmol) and a solution of acrylic acid in DMF (25 µl
× 4.4 M, 0.09 mmol) were added. Encapsulated Pd catalyst
(Pd–EnCat, 6 mg, 3 mol%) was added and the reaction mix-
ture was stirred at 90 ЊC for 18 h. The mixture was cooled,
filtered and the supported catalyst was washed with DMF
(200 µl). To the combined filtrates were added EDC (22 mg,
0.12 mmol) and HOBt (18 mg, 0.14 mmol). The resulting mix-
ture was stirred at room temperature for 20 min when a solution
of o-(tetrahydro-2H-pyran-2-yl)hydroxylamine (16 mg, 0.14
mmol) in DMF (100 µl) was added and the reaction mixture
N-(Tetrahydro-pyran-2-yloxy)-3-[4-(toluene-4-sulfonylamino)-
phenyl]-acrylamide (11). To a solution of 3-[4-(toluene-4-
sulfonylamino)-phenyl]-acrylic acid 9 (66 mg, 0.20 mmol) in
DMF (1 ml), was added a solution of diethylaminopropyl-
carbodiimide hydrochloride (EDC) (50 mg, 0.30 mmol) and
1-hydroxybenzotriazole (HOBt) (40.5 mg, 0.30 mmol) in DMF
(1.5 ml). The reaction mixture was stirred at room temperature
for 20 min when a solution of o-(tetrahydro-2H-pyran-2-yl)-
hydroxylamine (35 mg, 0.30 mmol) in DMF (500 µl) was added.
The resulting mixture was heated at 50 ЊC for 24 h. Upon
cooling to room temperature, the reaction mixture was applied
to a NH2-SPE cartridge (2 g) and eluted with methanol to
afford the tetrahydropyranyl ether 11 as an off-white solid
(49 mg, 70%). Rf = 0.25 [EtOAc/hexane (1 : 1)]; HRMS:
C21H24N2O5S requires (MH)ϩ 417.1484, found 417.1501; 1H
NMR (400 MHz, CD3OD): δ 7.62 (d, 1H, J = 8 Hz), 7.49 (d,
2H, J = 16 Hz), 7.38 (d, 2H, J = 8 Hz), 7.27 (d, 2H, J = 8 Hz),
7.10 (d, 2H, J = 8 Hz), 6.36 (d, 1H, J = 16 Hz), 4.94 (br s, 1H),
4.01 (t, 1H, J = 8 Hz), 3.61 (m, 1H), 3.34 (s, 3H), 2.35 (s, 3H),
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 1 1 – 6 2 0
616

View Article Online
heated at 50 ЊC for 18 h. The resulting mixture was applied to a
NH2-SPE cartridge (1 g) and eluted with methanol. Amberlyst
H-15 resin (70 mg) was added to the collected eluate and
the suspension was agitated at rt for 2 h. The resin was then
drained, washed with methanol (0.5 ml × 2) and the combined
filtrates were evaporated in vacuo to leave the hydroxamic acid
(NB: For the following examples overall yields are quoted for
the entire sequence after HPLC purification).
N-Hydroxy-3-[4-(toluene-4-sulfonylamino)-phenyl]acrylamide
(1a). White solid (11 mg, 26%). Data as previously.
1
1a as an off-white solid (20 mg, 45%); H NMR (400 MHz,
N-Hydroxy-3-{4-[methyl-(toluene-4-sulfonyl)-amino]-phenyl}-
acrylamide (1b). White solid (10.5 mg, 23%). Data as previously.
CD3OD): δ 7.65 (d, 2H, J = 8 Hz), 7.46 (d, 1H, J = 16 Hz), 7.40
(d, 2H, J = 8 Hz), 7.28 (d, 2H, J = 8 Hz), 7.10 (d, 2H, J = 8 Hz),
2.34 (s, 3H); HPLC: tR = 3.92 min (87%; 254 nm). Further
purification by HPLC gave 1a as a white solid (10.5 mg, 24%):
HPLC: tR = 3.87 min (>98%; 254 nm). Data as previously.
N-Hydroxy-3-[3-(toluene-4-sulfonylamino)-phenyl]-acryl-
amide (1c). White solid (11 mg, 25%). IR νmax/cmϪ1: 1658
(C᎐O), 1603 (C᎐C), 1330 (SO ), 1151 (SO ); HRMS: C H -
᎐
᎐
2
2
16 16
N2O4S requires (MH)ϩ 333.0909, found 333.0914; H NMR
(500 MHz, CD3OD): δ 7.63 (d, 2H, J = 8 Hz), 7.44 (d, 1H,
J = 16 Hz), 7.30–7.20 (m, 5H), 7.06 (m, 1H), 6.38 (d, 1H,
J = 16 Hz), 2.01 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 168.3,
147.5, 143.2, 142.1, 140.3, 139.5, 133.0, 132.9, 130.6, 127.2,
125.5, 123.1, 121.5, 23.7; LC-MS: (ESI Ϫve) m/z 331 (MH)Ϫ, tR
= 2.72 min; HPLC: tR = 3.99 min (100%; 254 nm).
1
General procedure for the preparation of polystyrene-
supported DMAP:sulfonyl chloride adducts (5). Dried PS-
DMAP resin (600 mg, 3.0 mmol gϪ1, 1.80 mmol) and the sulfo-
nyl chloride (Table 1: 1.80 mmol) were suspended in anhydrous
CH2Cl2 (10 ml) and shaken for 1 h. The resin was filtered,
washed with CH2Cl2 (10 ml × 3) and dried in vacuo to afford the
PS-DMAP:sulfonyl chloride adduct 5.
Resin loadings were calculated from the mass of the sulfonyl
chloride recovered from the combined resin washings (typical
loading: 1.9 mmol gϪ1–2.2 mmol gϪ1).
N-Hydroxy-3-{3-[methyl-(toluene-4-sulfonyl)-amino]-phenyl}-
acrylamide (1d). Colourless gum (9.0 mg, 20%). IR νmax/cmϪ1
:
1659 (C᎐O), 1622 (C᎐O), 1599 (C᎐C), 1344 (SO ), 1165
᎐
᎐
᎐
2
(SO2); HRMS: C13H12N2O4S requires (MH)ϩ 347.1066, found
347.1057; 1H NMR (500 MHz, CD3OD): δ 7.51–7.38 (m,
4H), 7.35–7.29 (m, 4H), 7.06 (d, 1H, J = 8 Hz), 6.41 (d, 1H,
J = 16 Hz), 3.16 (s, 3H), 2.40 (s, 3H); 13C NMR (100 MHz,
CD3OD) δ 165.9, 145.6, 143.7, 140.6, 137.1, 134.6, 130.6, 130.5,
129.0, 128.6, 127.7, 126.9, 119.5, 38.5, 21.5; LC-MS: (ESI Ϫve)
m/z 345 (MH)Ϫ, tR = 2.98 min; HPLC: tR = 4.26 min (98%; 254
nm).
General procedure for the preparation of sulfonamides (6). To
the PS-DMAP:sulfonyl chloride adducts 5 (0.26 mmol) was
added a solution of an iodoaniline 4 (Table 1: 57 mg, 0.26
mmol) in DMF (2 ml). The resulting suspensions were stirred at
50 ЊC for 1 h, drained, and the resins washed with DMF
(0.5 ml). In each case a further quantity of PS-DMAP:sulfonyl
chloride resin (0.26 mmol) was added to the collected filtrates
and the mixtures were stirred at 40 ЊC for a further 1 h. Upon
cooling to rt, the suspensions were drained and the resins
washed with DMF (0.5 ml). The filtrates were applied to SCX
cartridges (1 g) and these were eluted with DMF (1 ml) to
afford solutions of the sulfonamides 6.
N-Hydroxy-3-[4-(toluene-3-sulfonylamino)-phenyl]-acryl-
amide (1e). White solid (10 mg, 24%). IR νmax/cmϪ1: 1678
(C᎐O), 1654 (C᎐O), 1598 (C᎐C), 1330 (SO ), 1146 (SO );
᎐
᎐
᎐
2
2
HRMS: C16H16N2O4S requires (MH)ϩ 333.0909, found
333.0922; 1H NMR (500 MHz, CD3OD): δ 7.60 (s, 1H), 7.58 (d,
1H, J = 7 Hz), 7.45 (d, 1H, J = 16 Hz), 7.41–7.32 (m, 4H), 7.05
(d, 2H, J = 8 Hz), 6.32 (d, 1H, J = 16 Hz), 2.34 (s, 3H); 13C
NMR (125 MHz, CD3OD): δ 166.0, 140.8, 140.6, 140.5, 134.7,
134.6, 132.2, 130.0, 129.7, 128.4, 125.3, 121.5, 117.5, 21.2;
LC-MS: (ESI Ϫve) m/z 331 (MH)Ϫ, tR = 2.65 min; HPLC:
tR = 3.79 min (96%; 254 nm).
General procedure for the preparation of methyl sulfonamides
(8). Half of each DMF solution of the sulfonamides 6 (1.5 ml)
from the previous step were added to suspensions of PS-BEMP
(45 mg, 0.10 mmol) in a solution of iodomethane (25 µl ×
4.4 M, 0.11 mmol) in DMF. The resulting mixtures were
agitated at rt for 18 h, then drained and the resins washed
with DMF (0.2 ml) to afford DMF solutions of the methyl
sulfonamides 8. These were used directly in the next step.
N-Hydroxy-3-{4-[methyl-(toluene-3-sulfonyl)-amino]-phenyl}-
acrylamide (1f ). Colourless gum (5.7 mg, 13%). IR νmax/cmϪ1
:
General procedure for Heck olefinations to prepare (9) and
(10). To the DMF solutions of the sulfonamides 6 or 8 (1.5 ml
or 1.7 ml respectively) was added triethylamine (35 µl, 0.35
mmol) followed by a solution of acrylic acid in DMF (25 µl ×
3.4 M; 0.09 mmol). Encapsulated Pd catalyst (Pd EnCat,
6 mg, 3 mol%) was added and the mixtures stirred at 85 ЊC for
18 h. The reaction mixtures were cooled, filtered and the pal-
ladium catalyst washed with DMF (200 µl). These solutions
were used directly in the next step.
1659 (C᎐O), 1625 (C᎐O), 1603 (C᎐C), 1348 (SO ), 1150 (SO );
᎐ ᎐ ᎐
2 2
HRMS: C17H18N2O4S requires (MH)ϩ 347.1066, found
347.1080; 1H NMR (500 MHz, CD3OD): δ 7.57–7.29 (m, 6H),
7.14 (d, 2H), 6.44 (d, 1H, J = 16 Hz), 3.17 (s, 3H), 2.34 (s, 3H);
13C NMR (500 MHz, CD3OD): δ 166.0, 144.1, 140.6, 140.4,
137.8, 135.1, 135.0, 130.0, 129.2, 129.1, 127.8, 126.0, 119.2,
38.3, 21.2; LC-MS: (ESI Ϫve) m/z 345 (MH)Ϫ, tR = 2.84 min;
HPLC: tR = 4.26 min (100%; 254 nm).
N-Hydroxy-3-[3-(toluene-3-sulfonylamino)-phenyl]-acryl-
General procedure for the preparation hydroxamic acids
(1a–x). To the DMF solutions of the acrylic acids 9 and 10 in
DMF (1.5 ml) was added EDC (22 mg, 0.10 mmol) and HOBt
(18 mg, 0.13 mmol). The reaction mixtures were stirred at room
temperature for 20 min. A solution of o-(tetrahydro-2H-pyran-
2-yl)hydroxylamine (10.5 mg, 0.09 mmol) in DMF (100 µl) was
added and the resulting mixtures were gently stirred at 50 ЊC for
18 h. The solutions were applied to NH2-SPE cartridges (1 g)
and the cartridges were eluted with methanol. Amberlyst H–15
resin (70 mg) was added to the column eluates and the resulting
suspensions were agitated at rt for 2 h. The resins were drained,
washed with methanol (0.5 ml × 2) and the combined filtrates
were evaporated in vacuo. The products obtained were purified
by HPLC to gave the hydroxamic acids 1a–x.
amide (1g). White solid (10.5 mg, 24%). IR νmax/cmϪ1: 1661
(C᎐O), 1603 (C᎐C), 1332 (SO ), 1150 (SO ); HRMS: C H -
᎐
᎐
2
2
16 16
N2O4S requires (MH)ϩ 333.0909, found 333.0909; H NMR
(500 MHz, CD3OD): δ 7.59 (m, 2H), 7.44 (d, 1H, J = 16 Hz),
7.38–7.21 (m, 5H), 7.06 (m, 1H), 6.39 (d, 1H, J = 16 Hz), 2.34 (s,
3H); 13C NMR (125 MHz, CD3OD): δ 166.4, 141.4, 141.3,
140.2, 137.6, 135.1, 131.1, 130.3, 128.8, 125.7, 125.4, 123.7,
121.2, 119.6, 21.6; LC-MS: (ESI Ϫve) m/z 331 (MH)Ϫ, tR = 2.69
min; HPLC: tR = 3.88 min (100%; 254 nm).
1
N-Hydroxy-3-{3-[methyl-(toluene-3-sulfonyl)-amino]-phenyl}-
acrylamide (1h). Colourless gum (8.8 mg, 20%). IR νmax/cmϪ1
:
1662 (C᎐O), 1625 (C᎐O), 1600 (C᎐C), 1348 (SO ), 1149
᎐
᎐
᎐
2
(SO2); HRMS: C17H18N2O4S requires (MH)ϩ 347.1066, found
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 1 1 – 6 2 0
617

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347.1075; 1H NMR (500 MHz, CD3OD): δ 7.51–7.28 (m, 8H),
7.08 (m, 1H), 6.40 (d, 1H, J = 16 Hz), 3.17 (s, 3H), 2.34 (s, 3H);
13C NMR (125 MHz, CD3OD): δ 165.9, 143.7, 140.6, 140.5,
137.4, 137.1, 135.0, 130.5, 129.9, 129.2, 128.7, 127.8, 126.9,
126.1, 119.5, 38.5, 21.2; LC-MS: (ESI Ϫve) m/z 345 (MH)Ϫ,
tR = 2.82 min; HPLC: tR = 4.19 min (100%; 254 nm).
7H), 7.16 (d, 2H, J = 9 Hz), 6.46 (d, 1H, J = 16 Hz), 3.19 (s, 3H);
13C NMR (125 MHz, CD3OD): δ 166.1, 143.8, 140.7, 136.3,
135.4, 130.5, 130.4, 129.4, 127.9, 119.4, 38.5; LC-MS: (ESI
Ϫve) m/z 365 (MH)Ϫ, tR = 2.91 min; HPLC: tR = 4.48 min
(100%; 254 nm).
3-[3-(4-Chloro-benzenesulfonylamino)-phenyl]-N-hydroxy-
3-[4-(Biphenyl-4-sulfonylamino)-phenyl]-N-hydroxy-acryl-
acrylamide (1o). Colourless gum (9.8 mg, 21%). IR νmax/cmϪ1
:
amide (1i). White solid (3.2 mg, 6%). IR νmax/cmϪ1: 1676 (C᎐O),
1661 (C᎐O), 1590 (C᎐C), 1338 (SO ), 1153 (SO ); HRMS:
᎐ ᎐
2 2
1
᎐
1654 (C᎐O), 1333 (SO ), 1155 (SO ); HRMS: C H N O S
C15H13N2O4ClS requires (MH)ϩ 353.0363, found 353.0360; H
NMR (500 MHz, CD3OD): δ 7.72 (d, 2H, J = 8 Hz), 7.54–
7.41 (m, 3H), 7.32–7.22 (m, 3H), 7.05 (m, 1H), 6.40 (d, 1H,
J = 16 Hz); 13C (125 MHz, CD3OD): δ 166.0, 140.8, 140.3,
139.7, 139.5, 137.4, 130.9, 130.4, 129.9, 125.3, 123.5, 121.2,
119.4; LC-MS: (ESI Ϫve) m/z 351 (MH)Ϫ, tR = 2.80 min;
HPLC: tR = 3.98 min (100%; 254 nm).
᎐
2
2
21 18
2
4
requires (MH)ϩ 395.1066, found 395.1080; 1H NMR (500
MHz, CD3OD): δ 7.85 (d, 2H, J = 8 Hz), 7.73 (d, 2H, J = 8 Hz),
7.61 (d, 2H, J = 8 Hz), 7.48–7.35 (m, 6H), 7.16 (d, 2H, J = 8 Hz,
H), 6.33 (d, 1H, J = 16 Hz); 13C NMR (125 MHz, CD3OD):
δ 166.7, 146.0, 139.8, 139.3, 138.0, 131.3, 128.8, 128.5, 128.3,
127.5, 127.4, 127.3, 127.0, 120.4; LC-MS: (ESI Ϫve) m/z 393
(MH)Ϫ, tR = 3.01 min; HPLC: tR = 4.58 min (97%; 254 nm).
3-{3-[(4-Chloro-benzenesulfonyl)-methyl-amino]-phenyl}-N-
3-{4-[(Biphenyl-4-sulfonyl)-methyl-amino]-phenyl}-N-hydroxy-
acrylamide (1j). White solid (8.0 mg, 16%). IR νmax/cmϪ1: 1651
(C᎐O), 1605 (C᎐O), 1597 (C᎐C), 1345 (SO ), 1153 (SO ); HRMS:
hydroxy-acrylamide (1p). White solid (9.7 mg, 20%). IR νmax/
cmϪ1: 1660 (C᎐O), 1628 (C᎐O), 1602 (C᎐C), 1343 (SO ), 1164
᎐
᎐
᎐
2
(SO2); HRMS: C16H15ClN2O4S requires (MH)ϩ 367.0519,
᎐
᎐
᎐
2
2
1
C22H20N2O4S requires (MH)ϩ 409.1222, found 409.1239; 1H
NMR (500 MHz, CD3OD): δ 7.78 (d, 2H, J = 8 Hz), 7.67 (d, 2H,
J = 8 Hz), 7.60 (d, 2H, J = 8 Hz), 7.54–7.44 (m, 5H), 7.40 (m, 1H),
found 367.0526; H NMR (125 MHz, CD3OD): δ 7.52–7.46
(m, 6H), 7.35 (m, 2H), 7.08 (dd, 1H, J = 1.5, 9 Hz), 6.43 (d,
1H, J = 16 Hz), 3.20 (s, 3H); 13C NMR (125 MHz, CD3OD):
δ 166.0, 143.4, 140.7, 140.5, 137.3, 136.4, 130.6, 130.4, 128.6,
127.8, 127.1, 119.7, 38.6; LC-MS: (ESI Ϫve) m/z 365 (MH)Ϫ, tR
= 2.89 min; HPLC: tR = 4.45 min (100%; 254 nm).
7.20 (d, 2H, J = 8 Hz), 6.44 (d, 1H, J = 16 Hz), 3.22 (s, 3H); 13
C
NMR (125 MHz, CD3OD) δ 147.3, 144.1, 140.4, 140.3, 136.2,
135.2, 130.2, 129.8, 129.6, 129.5, 129.3, 128.5, 128.3, 127.8, 127.0,
119.2; LC-MS: (ESI Ϫve) m/z 407 (MH)Ϫ, tR = 3.19 min; HPLC:
tR = 5.12 min (100%; 254 nm).
3-[4-(3,4-Dimethoxy-benzenesulfonylamino)-phenyl]-N-
hydroxy-acrylamide (1q). Colourless gum (11 mg, 23%). IR νmax
/
3-[3-(Biphenyl-4-sulfonylamino)-phenyl]-N-hydroxy-acryl-
amide (1k). Colourless gum (9.8 mg, 19%). IR νmax/cmϪ1: 1660
(C᎐O), 1596 (C᎐C), 1331 (SO ), 1154 (SO ); HRMS C H -
cmϪ1: 1665 (C᎐O), 1603 (C᎐C), 1508 (Ar–H), 1332 (SO ), 1262
᎐
᎐
2
(C–O), 1136 (SO2); HRMS: C17H18N2O6S requires (MH)ϩ
379.0964, found 379.0975; 1H NMR (500 MHz, CD3OD):
δ 7.45–7.35 (m, 4H), 7.24 (s, 1H), 7.12 (d, 2H, J = 8 Hz), 6.97 (d,
1H, J = 8 Hz), 6.34 (d, 1H, J = 16 Hz), 3.82 (s, 3H), 3.76 (s, 3H);
13C NMR (125 MHz, CD3OD): 166.7, 154.7, 150.6, 141.2,
132.8, 132.7, 130.1, 122.8, 122.2, 118.0, 112.3, 114, 57.0 (×2);
LC-MS: (ESI Ϫve) m/z 377 (MH)Ϫ, tR = 2.46 min; HPLC:
tR = 3.34 min (95%; 254 nm).
᎐
᎐
2
2
21 18
N2O4S requires (MH)ϩ 395.1066, found 395.1075; H NMR
(500 MHz, CD3OD): δ 7.82 (d, 2H, J = 8 Hz), 7.71 (d, 2H,
J = 8 Hz), 7.61 (d, 2H, J = 8 Hz), 7.48–7.33 (m, 5H), 7.24 (m,
2H), 7.11 (m, 1H), 6.39 (d, 1H, J = 16 Hz); 13C NMR (125
MHz, CD3OD): δ 164.4, 146.9, 141.4, 141.0, 140.2, 140.1,
137.2, 131.2, 130.5, 129.9, 129.2, 129.0, 128.7, 125.5, 123.7,
121.3, 119.7; LC-MS: (ESI Ϫve) m/z 393 (MH)Ϫ, tR = 3.07 min;
HPLC: tR = 4.02 min (100%; 254 nm).
1
3-{4-[(3,4-Dimethoxy-benzenesulfonyl)-methyl-amino]-
phenyl}-N-hydroxy-acrylamide (1r). Colourless gum (8.8 mg,
17%). HRMS: C18H20N2O6S requires (MH)ϩ 393.1120, found
3-{3-[(Biphenyl-4-sulfonyl)-methyl-amino]-phenyl}-N-hydroxy-
acrylamide (1l). White solid (4.5 mg, 8%). IR νmax/cmϪ1: 1656
1
393.1137; H NMR (CD3OD): δ 7.56–7.49 (m, 3H), 7.19 (dd,
(C᎐O), 1604 (C᎐O), 1596 (C᎐C), 1346 (SO ), 1163 (SO ); HRMS:
1H, J = 8, 1 Hz), 7.17 (d, 2H, J = 8 Hz), 7.05 (d, 1H, J = 8 Hz),
6.83 (d, 1H, J = 1 Hz), 6.45 (d, 1H, J = 16 Hz), 3.88 (s, 3H), 3.65
(s, 3H), 3.15 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 166.1,
154.7, 150.3, 144.4, 140.4, 135.1, 129.1, 128.9, 127.8, 123.0,
119.2, 112.0, 111.7, 56.6, 56.4, 38.2; LC-MS: (ESI Ϫve) m/z 391
(MH)Ϫ, tR = 2.62 min; HPLC: tR = 3.83 min (100%; 254 nm).
᎐
᎐
᎐
2
2
C22H20N2O4S requires (MH)ϩ 409.1222, found 409.1234; 1H
NMR (500 MHz, CD3OD): δ 7.85 (d, 2H, J = 8 Hz), 7.66 (d, 2H,
J = 8 Hz), 7.60 (d, 2H, J = 8 Hz), 7.52–7.41 (m, 4H), 7.38–7.32 (m,
3H), 7.12 (d, 1H, J = 8 Hz), 6.42 (d, 1H, J = 16 Hz), 3.25 (s, 3H);
13C NMR (125 MHz, CD3OD): δ 166.0, 147.3, 143.7, 140.6,
137.2, 136.2, 130.6, 130.2, 129.7, 129.6, 128.7, 128.5, 128.3, 127.8,
127.0, 119.6, 38.6; LC-MS: (ESI Ϫve) m/z 407 (MH)Ϫ, tR = 3.19
min; HPLC: tR = 5.12 min (100%; 254 nm).
3-[3-(3,4-Dimethoxy-benzenesulfonylamino)-phenyl]-N-
hydroxy-acrylamide (1s). White solid (12 mg, 24%). IR νmax
/
cmϪ1: 1658 (C᎐O), 1588 (C᎐C), 1509 (Ar–H), 1332 (SO ), 1264
᎐
᎐
2
3-[4-(4-Chloro-benzenesulfonylamino)-phenyl]-N-hydroxy-
(C–O), 1140 (SO2); HRMS: C17H18N2O6S requires (MH)ϩ
379.0964, found 379.0977; 1H NMR (500 MHz, CD3OD):
δ 7.46 (d, 1H, J = 16 Hz), 7.38–7.28 (m, 2H), 7.26–7.19 (m, 3H),
7.06 (m, 1H), 6.97 (d, 1H, J = 8 Hz), 6.40 (d, 1H, J = 16 Hz),
3.84 (s, 3H), 3.75 (s, 3H); 13C NMR (125 MHz, CD3OD):
δ 166.5, 154.7, 150.6, 141.3, 140.4, 137.6, 132.6, 131.1, 125.4,
123.9, 122.8, 121.5, 119.6, 112.3, 111.4, 57.0 (×2); LC-MS: (ESI
Ϫve) m/z 377 (MH)Ϫ, tR = 2.50 min; HPLC: tR = 3.43 min (95%;
254 nm).
acrylamide (1m). White solid (4.4 mg, 10%). IR νmax/cmϪ1: 1654
(C᎐O), 1599 (C᎐C), 1337 (SO ), 1162 (SO ); HRMS: C H -
᎐
᎐
2
2
15 13
N2O4ClS requires (MH)ϩ 353.0363, found 353.0363; H NMR
(500 MHz, CD3OD): δ 7.74 (d, 2H, J = 8 Hz), 7.52–7.39 (m,
5H), 7.1 (d, 2H, J = 8 Hz), 6.34 (d, 1H, J = 16 Hz); 13C NMR
(125 MHz, CD3OD): δ 166.4, 140.7, 140.3, 140.2, 139.8, 132.6,
130.4, 129.9, 129.8, 121.9, 117.8; LC-MS: (electrospray Ϫve)
m/z 351 (MH)Ϫ, tR = 2.76 min; HPLC: tR = 3.98 min (95%; 254
nm).
1
3-{3-[(3,4-Dimethoxy-benzenesulfonyl)-methyl-amino]-
phenyl}-N-hydroxy-acrylamide (1t). White solid (9.8 mg, 19%).
IR νmax/cmϪ1: 1662 (C᎐O), 1625 (C᎐O), 1588 (C᎐C), 1508
3-{4-[(4-Chloro-benzenesulfonyl)-methyl-amino]-phenyl}-N-
hydroxy-acrylamide (1n). Colourless gum (8.7 mg, 18%). IR
νmax/cmϪ1: 1659 (C᎐O), 1625 (C᎐O), 1604 (C᎐C), 1352 (SO ),
᎐
᎐
᎐
(Ar–H), 1347 (SO2), 1261 (C–O), 1137 (SO2); HRMS: C18H20-
᎐
᎐
᎐
2
1
1173 (SO2); HRMS: C16H15ClN2O4S requires (MH)ϩ 367.0519,
N2O6S requires (MH)ϩ 393.1120, found 393.1129; H NMR
found 367.0532; 1H NMR (500 MHz, CD3OD): δ 7.56–7.49 (m,
(500 MHz, CD3OD): δ 7.53–7.46 (m, 2H), 7.37–7.33 (m, 2H),
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 1 1 – 6 2 0
618

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7.21 (dd, 1H, J = 1, 8 Hz), 7.11 (d, 1H, J = 8 Hz), 7.06 (d,
1H, J = 8 Hz), 6.83 (d, 1H, J = 1 Hz), 6.43 (d, 1H, J = 16 Hz),
3.88 (s, 3H), 3.65 (s, 3H), 3.16 (s, 3H); 13C NMR (125 MHz,
CD3OD): δ 165.9, 154.7, 150.3, 143.9, 140.6, 137.1, 130.5,
128.8, 128.7, 127.7, 126.9, 123.1, 119.5, 112.0, 111.8, 56.7, 56.5,
38.4; LC-MS: (ESI Ϫve) m/z 391 (MH)Ϫ, tR = 2.63 min; HPLC:
tR = 3.78 min (100%; 254 nm).
supplemented with 10% foetal bovine serum (FBS) (Invitro-
gen), 1% penicillin/streptomycin, 30 µg mlϪ1 endothelial cell
growth supplement (ECGS) from bovine neural tissue and
90 µg mlϪ1 bovine intestinal mucosal heparin (Sigma). The
cells were seeded in tissue culture-ware coated with 1.5%
gelatin. HUVECs were used between passage 2 and 7. For
the proliferation assay, four thousand cells were plated on
gelatin-coated 96-well plates in 1% FBS-containing medium
and allowed to adhere overnight. Serial dilutions of test com-
pounds were added and cells were incubated at 37 ЊC, 5% CO2
in a humidified incubator. Cell number was determined using
the CellTiter 96® AQueous One Solution Reagent (Promega) at
the end of 48 h by reading optical density/absorbance at 492
nm.
N-Hydroxy-3-[4-(thiophene-2-sulfonylamino)-phenyl]-acryl-
amide (1u). Colourless gum (7.8 mg, 18%). IR νmax/cmϪ1: 1655
(C᎐O), 1604 (C᎐C), 1336 (SO2), 1151 (SO2); HRMS:
᎐
᎐
C13H12N2O4S requires (MH)ϩ 325.0317, found 325.0332; 1H
NMR (500 MHz, CD3OD): δ 7.70 (dd, 1H, J = 5, 1.5 Hz),
7.52 (dd, 1H, J = 4, 1.5 Hz), 7.50–7.42 (m, 3H), 7.17 (d, 2H,
J = 8 Hz), 7.06 (dd, 1H, J = 5, 4 Hz), 6.38 (d, 1H, J = 16 Hz);
LC-MS: (ESI Ϫve) m/z 323 (MH)Ϫ, tR = 2.48 min; HPLC:
tR = 3.64 min (100%; 254 nm).
AngioKit tube-formation assay. The AngioKit was purchased
from TCS Cellworks.27 Test compounds were dissolved in
DMSO and diluted in the optimised medium provided with the
kit such that the final DMSO concentration was always below
0.1%. The medium was replaced on day 1, 4, 7, and 9 with fresh
medium containing appropriate compounds. Suramin (2 µM)
containing medium provided a negative control, and medium
without any compound was used as blank. Tubules were visual-
ized at the end of 11 days by immunostaining with CD31/
PECAM-1 antibodies (TCS). Primary antibody was diluted
1 : 4000 and secondary, goat anti-mouse IgG alkaline phos-
phatase conjugated antibody was diluted 1 : 500. Colour was
developed using BCIP/NBT (Sigma) and images captured with
a Nikon Diaphot microscope and analysed using AngioSys
image analysis software.
N-Hydroxy-3-{4-[methyl-(thiophene-2-sulfonyl)-amino]-
phenyl}-acrylamide (1v). White foam (8.4 mg, 19%). IR νmax
/
cmϪ1: 1655 (C᎐O), 1602 (C᎐C), 1349 (SO ), 1148 (SO ); HRMS:
᎐
᎐
2
2
C14H14N2O4S2 requires (MH)ϩ 339.0473, found 339.0473; H
NMR (CD3OD): δ 7.80 (dd, 1H, J = 5, 1.5 Hz), 7.56–7.49 (m,
3H), 7.38 (dd, 1H, J = 4, 1.5 Hz), 7.18 (d, 2H, J = 8 Hz), 7.15 (m,
1H), 6.39 (d, 1H, J = 16 Hz), 3.24 (s, 3H); 13C NMR (125 MHz,
CD3OD): δ 166.1, 143.8, 140.4, 137.4, 135.3, 134.3, 134.2,
129.2, 128.7, 127.9, 119.2, 38.5; LC-MS: (ESI Ϫve) m/z 337
(MH)Ϫ, tR = 2.67 min; HPLC: tR = 3.88 min (100%; 254 nm).
1
N-Hydroxy-3-[3-(thiophene-2-sulfonylamino)-phenyl]-acryl-
amide (1w). Colourless gum (8.9 mg, 21%). IR νmax/cmϪ1: 1654
Statistics. All assays were performed in triplicate. For the
HDAC enzyme inhibition assay, an average of three OD read-
ings at 405 nm were converted to the amount of deacetylated
substrate remaining, reading off from a pre-determined cali-
bration curve. The HUVEC proliferation assay was performed
in triplicate and statistical significance was calculated using
ANOVA followed by Tukey’s test. For the AngioKit assay 8
image fields were captured and the mean tubule area was calcu-
lated. Values were considered statistically significant at P ≤ 0.05.
(C᎐O), 1615 (C᎐O), 1604 (C᎐C), 1326 (SO ), 1147 (SO );
᎐
᎐
᎐
2
2
HRMS: C13H12N2O4S requires (MH)ϩ 325.0317, found
325.0323; 1H NMR (500 MHz, CD3OD): δ 7.70 (dd, 1H, J = 5,
1.5 Hz), 7.50 (dd, 1H, J = 4, 1.5 Hz), 7.46 (d, 1H, J = 16 Hz),
7.34 (br s, 1H), 7.30–7.28 (m, 2H), 7.12 (m, 1H), 7.04 (dd, 1H,
J = 5, 4 Hz) 6.40 (d, 1H, J = 16 Hz); LC-MS: (ESI Ϫve) m/z 323
(MH)Ϫ, tR = 2.51 min; HPLC: tR = 3.50 min (100%; 254 nm).
N-Hydroxy-3-{3-[methyl-(thiophene-2-sulfonyl)-amino]-
phenyl}-acrylamide (1x). Colourless gum (7.3 mg, 17%). IR
νmax/cmϪ1: 1662 (C᎐O), 1625 (C᎐O), 1351 (SO ), 1165 (SO );
Acknowledgements
᎐
᎐
2
2
HRMS: C14H14N2O4S2 requires (MH)ϩ 339.0473, found
339.0467; 1H NMR (500 MHz, CD3OD): δ 7.82 (dd, 1H, J = 5,
1.5 Hz), 7.55–7.46 (m, 3H), 7.22–7.08 (m, 4H), 6.42 (d, 1H,
J = 16 Hz), 3.25 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 165.9,
143.4, 140.5, 134.3, 134.2, 130.6, 128.8, 128.7, 128.6, 128.0,
126.9, 119.6, 38.6; LC-MS: (ESI Ϫve) m/z 337 (MH)Ϫ, tR = 2.65
min; HPLC: tR = 3.84 min (100%; 254 nm).
We thank GlaxoSmithKline for generous financial support of
this work, The Cambridge Commonwealth Trust and the
Department of Pharmacology for funding to AB. Gabrielle
Bradfield and Colin Chan are thanked for assistance with the
biological screening.
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27 AngioKit
was
obtained
from
TCS
Cellworks
(www.tcscellworks.co.uk).
28 Greenhouse parallel reaction block commercially available from
Radleys Discovery Technology catalogue no: RR99600
(www.radleys.co.uk).
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part numbers 2476000 and 0568900 respectively.
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620