Identification and optimisation of a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids as potent histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.10.048

Optimisation of ADS100380, a sub-micromolar HDAC inhibitor identified using a virtual screening approach, led to a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids (6a-i), that possessed significant HDAC inhibitory activity. Subseque

Full text of DOI:10.1016/j.bmcl.2006.10.048

Bioorganic & Medicinal Chemistry Letters 17 (2007) 370–375
Identification and optimisation of a series of substituted
5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids as potent
histone deacetylase (HDAC) inhibitors
Steve Price,* Walter Bordogna, Richard J. Bull, David E. Clark, Peter H. Crackett,
Hazel J. Dyke, Matthew Gill, Neil V. Harris, Julia Gorski, Julia Lloyd, Peter M. Lockey,
Julia Mullett, Alan G. Roach, Fabien Roussel and Anne B. White
Argenta Discovery Ltd, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, UK
Received 25 August 2006; revised 19 October 2006; accepted 20 October 2006
Available online 24 October 2006
Abstract—Optimisation of ADS100380, a sub-micromolar HDAC inhibitor identified using a virtual screening approach, led to a
series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids (6a–i), that possessed significant HDAC inhibitory activity.
Subsequent functionalisation of the pendent phenyl group of compounds 6f and 6g provided analogues 6j–w with further enhanced
enzyme and anti-proliferative activity. Compound 6j demonstrated efficacy in a mouse xenograft experiment.
Ó 2006 Elsevier Ltd. All rights reserved.
The histone deacetylases (HDACs) belong to a family of
zinc-dependent enzymes that cleave acetyl groups
appended to the e-amino side chain of lysine residues,
such as those found in the amino-terminal tails of his-
tone proteins. Deacetylated histones acquire a net posi-
tive charge that interacts strongly with the negatively
charged DNA, which becomes tightly wound around
the nucleosome core. Therefore, changing the acetyla-
tion status of the histone tails can affect chromatin struc-
ture, DNA accessibility to transcription factors, and
ultimately gene expression. It is known that, in malig-
nant cells, epigenetic events such as histone deacetyla-
tion can contribute to the transformed phenotype, and
inhibition of HDAC enzymes has been shown to induce
growth arrest, differentiation and apoptosis through the
lides, electrophilic ketones and cyclic peptides.3 Howev-
er, the most extensively exemplified class of inhibitors
is based upon the hydroxamic acid moiety, which is
a widely recognized zinc-binding group.4 Figure 1
illustrates some typical hydroxamic acid-containing
HDAC inhibitors, including trichostatin A (TSA),5
NVP-LAQ824,6 PXD101,7 and SAHA (ZolinzaTM, Vori-
nostat),8 which has been recently approved by the FDA
for once-daily oral treatment of advanced cutaneous
T-cell lymphoma (CTCL).
At Argenta, we initiated a hit-finding exercise that
utilised a virtual screening approach based on the
published crystal structure (PDB code: 1C3R) of
HDAC-like protein (HDLP).9 A virtual library of
increased expression of tumour suppressor genes such as
p21WAF1/CIP1
1
.
O
O
OH
O
OH
OH
N
H
N
H
Several different classes of HDAC inhibitors have en-
tered clinical trials, some of which have demonstrated
significant anti-proliferative activity against a number
of solid and haematological tumours.2 These classes of
HDAC inhibitors include aliphatic acids, o-aminoani-
N
N
N
H
TSA
NVP-LAQ824
O
O
H
O
O
N
OH
S
OH
N
H
N
N
H
H
O
PXD101
SAHA
Zolinza (Vorinostat)
Keywords: Histone deacetylase inhibitors; HDAC; Hydroxamic acids.
*
Corresponding author. Tel.: +44 1279 645622; fax: +44 1279
645646; e-mail: steve.price@argentadiscovery.com
Figure 1. Typical hydroxamic acid-containing HDAC inhibitors.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.048

S. Price et al. / Bioorg. Med. Chem. Lett. 17 (2007) 370–375
371
644 hydroxamic acids was generated from a data-
base of carboxylic acids available in-house, followed
by a docking and scoring exercise using the FlexX
program.10 Based on the virtual screening results,
75 compounds were synthesised and tested in an
HDAC enzyme assay,11 from which ADS100380
binds is located at the bottom of a tube-shaped pocket.
Thus, optimisation of ADS100380 was designed to
make additional interactions at the entrance to the
HDAC active site by tethering hydrophobic aromatic
groups to the pyrazole nitrogen (Scheme 1).13
(Fig. 2) was identified as a sub-micromolar (IC50
=
The commercially available vinylogous amide 1 was
treated with hydrazine hydrate to give the key cyanothi-
ophene intermediate 2. Compound 2 was converted to
the corresponding carbomethoxythiophene (3), via basic
hydrolysis of the nitrile group to provide the carboxylic
acid, followed by esterification. Both intermediates 2
and 3 underwent regioselective14 alkylation with alkyl
halides to provide the 1-N-alkylated cyanothiophenes
4a–f, and the 1-N-alkylated carbomethoxythiophenes 5
and 8, respectively. Hydrolysis of compounds 4a–f
provided the corresponding carboxylic acids, which
were then coupled with O-THP-protected hydroxyl-
amine, and subsequent acidic deprotection liberated
the desired final hydroxamic acids 6a–f. Compound 5
was treated with trifluoroacetic acid in dichloromethane
to liberate the terminal amino group, which was
then subjected to reductive alkylation conditions with
0.75 lM, Table 1) HDAC inhibitor. ADS100380 also
displayed weak functional activity (IC50 = 11.4 lM,
Table 1) in a cell-based proliferation assay.12 The
corresponding carboxylic acid of ADS100380 was
inactive.
The HDLP structure indicated that the catalytic zinc ion
to which the hydroxamic acid group of ADS1000380
F C
O
N
S
OH
N
N
H
ADS100380
Figure 2. An HDAC inhibitor identified by virtual screening.
Table 1. HDAC and anti-proliferative assay results for compounds 6a–i
O
N
S
R
NHOH
N
6
Compound
ADS100380
R
HDACa (IC50, lM)
Cell proliferationa
MCF-7 (IC50, lM)
MDA-MB231 (IC50,lM)
—
0.750
0.153
0.100
0.034
0.022
0.020
0.021
0.033
0.069
0.019
11.4
31.5
6a
6b
6c
6d
6e
6f
2.06
1.24
0.52
0.50
0.35
0.12
0.46
0.47
0.15
4.54
2.91
2.19
1.50
0.75
0.84
1.80
1.50
0.53
O
O
O
N
H
N
H
6g
6h
6i
N
H
N
a Values are means of two experiments.

372
S. Price et al. / Bioorg. Med. Chem. Lett. 17 (2007) 370–375
O
O
d
b, e, f
S
HN
S
N
S
N
S
a
CN
CN
CN
R
R
Me2N
NHOH
N
N
N
2
4a-f
6a-j
1
b, c
j, e, f or k
N
N
S
CO2Me
CO2Me
BocHN
N
N
h, i
g
l
5
HN
S
R1
N
S
CO2Me
CO2Me
N
R2/H
N
N
OMe
3
7
S
m, n
MeO
8
Scheme 1. Reagents and conditions: (a) EtOH, hydrazine hydrate (1.1 equiv), reflux, 16 h (89%); (b) 1 M NaOH, reflux, 2 h (52–97%); (c) MeOH,
i
concd HCl, reflux, 16 h (93%); (d) RX (1.2 equiv), K2CO3, DMF, 70 °C, 16 h (43–90%); (e) Pr2NEt (2.5 equiv), DMF, H2NOTHP (1.1 equiv),
HATU (1.1 equiv) (60–95%); (f) pTSA, MeOH (23–85%); (g) BocHN(CH2)2Br (1.1 equiv), K2CO3, DMF, 70 °C, 16 h (88%); (h) CF3CO2H, CH2Cl2;
(i) R1CHO (0.9 equiv), MeOH, 16 h, then NaBH4, 2 h (50–73% two steps); (j) LiOH, H2O, CH3CN, 16 h (quant); (k) NH2OHÆHCl (5 equiv), KOH,
MeOH, 16 h (25–65%); (l) (MeO)2CHCH2Br (1.2 equiv), K2CO3, DMF, 90 °C, 16 h (60%); (m) 1 M HCl, THF, lW 150 °C, 100 s (80%); (n)
R1R2NH (1.1 equiv), Na(OAc)3BH, CH2ClCH2Cl, 16 h (65–80%).
a variety of aromatic aldehydes to yield tethered
carbomethoxythiophenes 7. Acidic deprotection of the
dimethyl acetal 8 to liberate the corresponding aldehyde,
followed by reductive amination conditions using
tetrahydroisoquinoline, provided the desired intermedi-
ate carbomethoxythiophene 7 from which compound
6i was obtained. Conversion of the carbomethoxythi-
ophenes 7 to the desired hydroxamic acids 6g–j was
achieved by ester hydrolysis, followed by the same
coupling and deprotection conditions used for the
1-N-alkylated cyanothiophenes 4a–f, or directly by
treating with basic hydroxylamine.
pound 6g by a methylene group to furnish the pheneth-
ylamino-tethered analogue 6h resulted in reduced
activity in the HDAC assay, but no loss of potency in
the cellular assay. Cyclisation of the secondary amino
group of compound 6g onto the aromatic ring by an eth-
yl linker, compound 6i, provided a marginal increase in
activity in the HDAC assay, and a 3-fold increase in
activity in both cell proliferation assays.
Encouraged by the promising activity of the unsub-
stituted phenyl-tethered analogues 6a–i, we next focused
our attention on investigating the effect of functionalis-
ing the phenyl rings of compounds 6g, and 6f, respective-
ly. Table 2 includes a selection of compounds synthesised
to investigate aromatic ring substitution SAR.
All compounds were initially tested in a primary HDAC
enzyme assay,11 and those possessing sub-micromolar
activity were then simultaneously evaluated in MCF-7
and MDA-MB231 cell-based proliferation assays.12
The aminoethyl-tethered compounds 6j–r were pro-
duced using the existing synthetic methodology as out-
lined in Scheme 1. However, a modified synthetic
route was required to make further acetamide-tethered
hydroxamic acids that would enable the use of commer-
cially available aromatic amines (Scheme 2).13
The benzyl-tethered compound 6a possessed a 5-fold
increase in HDAC potency over ADS100380, both in
the HDAC and the MCF-7 cell proliferation assays.
Increasing the linker length of compound 6a by a
methylene group, compound 6b, led to a minor increase
in HDAC activity, whereas increasing the linker length
by two methylene units, compound 6c, provided more
than a 4-fold increase in activity. Both compounds 6b
and 6c display corresponding increases in activity in
the MCF-7 cell proliferation assays. Replacement of a
methylene group in the tether of compound 6c with an
oxygen atom, compound 6d, had little effect on the
activity. Homologation of the tether of compound 6d
with a methylene group, compound 6e, provided no
further increase in activity in the HDAC assay, but a
2-fold increase in anti-proliferative activity in the
MDA-MB231 cell-based assay. Introducing an acetam-
ide moiety between the phenyl and pyrazole ring sys-
tems, compound 6f, afforded the most potent three-
atom tether identified. Compound 6g was designed to
incorporate a basic amino group into a four-atom
tether, a transformation that retained similar activity
to the non-basic tethered analogue 6e in the HDAC
and MCF-7 assays. Homologation of the tether of com-
The carbomethoxythiophene 3 was alkylated with tert-
butyl bromoacetate to provide compound 9. The tert-
butyl ester 9 was readily hydrolysed and the resulting
acid was coupled with a variety of aromatic amines to
give compounds of the generic structure 10. Standard
conditions were then used to convert the methyl carbox-
ylate to the corresponding hydroxamic acids 6s–w.
The benzodioxole analogue 6j was one of the first substi-
tuted phenyl-tethered analogues to be prepared and test-
ed. Compound 6j indicated that phenyl substitution was
tolerated, and provided potent HDAC and cell prolifer-
ation IC50 values. The introduction of a para-methoxy
substituent to compound 6g afforded analogue 6k that
possessed a 3-fold increase in HDAC activity. However,
the 3,4-bis-methoxy substituted phenyl compound 6l
lost almost an order of magnitude in potency in both
HDAC and cellular proliferation assays. 5,6-Fused ring
systems attached either by the five- or the six-membered

S. Price et al. / Bioorg. Med. Chem. Lett. 17 (2007) 370–375
Table 2. HDAC and anti-proliferative assay results for compounds 6j–w
373
O
N
S
R
NHOH
N
6
Compound
R
HDACa (IC50, lM)
Cell proliferationa
MDA-MB231 (IC50,lM)
MCF-7 (IC50, lM)
O
N
H
6j
0.029
0.013
0.107
0.005
0.011
0.006
0.005
0.048
0.009
0.012
0.007
0.008
0.008
0.006
0.30
0.63
0.55
4.50
0.28
0.30
0.37
0.12
1.51
0.56
2.00
0.20
0.70
0.23
0.30
O
N
H
6k
6l
0.20
1.58
0.08
0.14
0.17
0.06
0.80
0.19
0.37
0.06
0.14
0.12
0.10
MeO
MeO
MeO
N
H
N
H
6m
6n
6o
6p
6q
6r
6s
6t
N
H
N
H
O
N
N
H
N
H
N
N
N
H
N
N
H
N
N
O
N
H
NH
O
N
H
N
F
O
6u
6v
6w
F
N
H
O
N
H
OM e
O
Cl
N
H
a Values are means of two experiments.
ring system, compounds 6m–o, provided very potent
compounds in the HDAC assay. Two heterobiaryl ring
systems, compounds 6p and 6r, possessing a terminal
heteroaromatic ring, had excellent activity. However,
the heterobiaryl ring system containing a terminal
phenyl ring system, compound 6q, displayed a much
reduced level of activity.
the HDAC assay, compounds 6s and 6t. Compounds
6u–w illustrate that, as for the ethylamino-tethered ser-
ies, substitution of the aromatic ring provides significant
potency in both the HDAC enzyme and the cellular
proliferation assays.
Further profiling of the substituted 5-(1H-pyrazol-3-yl)-
thiophene-2-hydroxamic acid series of compounds was
undertaken on one of the early phenyl substituted com-
pounds, 6j.
For the acetamide-tethered analogues, fused heteroaryl
ring systems once again provided significant activity in

374
S. Price et al. / Bioorg. Med. Chem. Lett. 17 (2007) 370–375
O
compound 6j in the mouse via an ip administration
route.
a
S
HN
N
S
CO2Me
CO2Me
t
BuO
N
N
3
9
Having achieved good plasma exposure for compound 6j
following ip dosing, we decided to undertake a xenograft
efficacy study using HCT116 tumour cells in CD1 mice.
Compound 6j was dosed to female CD1 nude (nu/nu)
athymic mice at doses of 5, 15 and 50 mg/kg, ip (n = 12
per group) for 21 days. The control vehicle was 10%
DMSO in arachis oil. This study resulted in a statistically
significant 33% (T/C = 0.67) tumour growth reduction
when compound 6j was dosed at 50 mg/kg by the ip
route. The tumours were removed 6 h after the last dose
of compound was administered. Significant levels of his-
tone acetylation were measured in tumours treated with
compound 6j compared to vehicle, indicating that
HDAC inhibition had occurred (data not shown).
b, c
O
O
d, e, f or g
N
S
N
S
CO2Me
R
ArHN
NHOH
N
N
6s-w
10
Scheme 2. Reagents and conditions: (a) tBuO2CCH2Br (1.1 equiv),
K2CO3, DMF, 80 °C, 16 h (90%); (b) CF3CO2H, Et3SiH, CH2Cl2
(75%); (c) Pr2Net (2.5 equiv), DMF, ArNH2 (1.1 equiv), HATU (1.1
i
i
equiv) (17–74%); (d) 1 M NaOH, reflux, 2 h (66%); (e) Pr2Net (2.5
equiv), DMF, H2NOTHP (1.1 equiv), HATU (1.1 equiv) (55%); (f)
p-TSA, MeOH (44%); (g) NH2OHÆHCl (5 equiv), KOH, MeOH, 16 h
(39–62%).
In summary, following the identification of ADS100380,
we have optimised a series of phenyl-tethered HDAC
inhibitors that display significant potency in in vitro en-
zyme and cell proliferation assays. Functionalisation of
the phenyl group of tethered 5-(1H-pyrazol-3-yl)-thio-
phene-2-hydroxamic acids 6g and 6f from this series
has led to compounds with improved potency, the best
of which possessed single digit nanomolar potency in
the HDAC enzyme assay, and sub-60 nanomolar activ-
ity in the MCF-7 cell proliferation assay. One of the first
phenyl-substituted compounds produced, 6j, demon-
strated an improved in vivo PK profile compared to
SAHA, and was also shown to be efficacious in a mouse
HCT116 xenograft study.
Table 3. HDAC and anti-proliferative assay results comparing com-
pound 6j and SAHA
Compound HDACa
Cell proliferationa (IC50, lM)
(IC50, lM)
MCF-7 MDA-
MB231
HCT116 PC3
SAHA
6j
0.125
0.029
1.50
0.30
12.3
0.63
2.52
0.58
3.11
0.40
a Values are means of two experiments.
Comparison of compound 6j with SAHA demonstrated
that 6j was 4-fold more potent than SAHA in the
primary HDAC enzyme assay, and had a 5- to 20-fold
improvement in anti-proliferative activity across a panel
of four cell lines (Table 3).
Acknowledgments
We would like to thank Mike Podmore and Russell
Scammell from the analytical department at Argenta
Discovery for NMR and LCMS analysis. We would
also like to thank Don Daley and Rob Jenkins from
the DMPK department at Argenta Discovery for the
in vivo studies completed on compounds within this
paper.
Compounds 6j and SAHA were subjected to rat (iv and
po, Table 4), and mouse (iv, po and ip, Table 5) PK
experiments. Compound 6j demonstrated reduced clear-
ance and increased half-life compared to SAHA in both
species. Poor oral bioavailability was observed for both
compounds in both species. However, excellent bioavail-
ability and good plasma exposure were observed for
Table 4. Rat PK profile for compound 6j and SAHA after iv (1 mg/kg) and po (5 mg/kg) dosing
Compound
t1/2 (h)
Vdss (L/kg)
CL (mL/min/kg)
iv
AUC0ꢀ1h
(lg h/mL)
po
F (%)
iv
iv
po
SAHA
6j
0.7
1.6
1.6
0.9
67
21
0.4
0.1
16
2
Table 5. Mouse PK profile for compound 6j and SAHA after iv (1 mg/kg), po (5 mg/kg) and ip (5 mg/kg) dosing
Compound
t1/2 (h)
Vdss (L/kg)
CL (mL/min/kg)
AUC0ꢀ1h
(lg h/mL)
F (%)
iv
iv
iv
po
ip
po
ip
SAHA
6j
0.8
2.5
2.1
0.4
78
10
0.2
0.9
0.6
7.2
14
10
61
85

S. Price et al. / Bioorg. Med. Chem. Lett. 17 (2007) 370–375
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2006.10.048.
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14. Regioselective alkylation was determined by 1H NMR
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4a and 4b.