Design and synthesis of thiazole-5-hydroxamic acids as novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.07.050

We have designed and synthesized a series of structurally novel hydroxamic acid-based histone deacetylase (HDAC) inhibitors characterized by a zinc chelating head group attached directly to a thiazole ring. The thiazole ring connects to a piperazine spacer, which is capped with a sulfonamide group. These novel molecules potently inhibit an HDAC enzyme mixture derived from HeLa cervical carcinoma cells and show potent antiproliferative activity against the breast cancer cell line MCF7.

Full text of DOI:10.1016/j.bmcl.2007.07.050

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5995–5999
Design and synthesis of thiazole-5-hydroxamic acids
as novel histone deacetylase inhibitors
Sampath-Kumar Anandan,*, John S. Ward,à Richard D. Brokx, Trisha Denny,
Mark R. Bray, Dinesh V. Patel and Xiao-Yi Xiao§
EntreMed Inc., 101 College Street, Toronto Medical Discovery Tower, Toronto, Ont., Canada M5G1L7
Received 13 April 2007; revised 14 July 2007; accepted 17 July 2007
Available online 19 August 2007
Abstract—We have designed and synthesized a series of structurally novel hydroxamic acid-based histone deacetylase (HDAC)
inhibitors characterized by a zinc chelating head group attached directly to a thiazole ring. The thiazole ring connects to a piperazine
spacer, which is capped with a sulfonamide group. These novel molecules potently inhibit an HDAC enzyme mixture derived from
HeLa cervical carcinoma cells and show potent antiproliferative activity against the breast cancer cell line MCF7.
Ó 2007 Elsevier Ltd. All rights reserved.
Epigenetics refers to the phenomenon in which gene
expression can be altered by reversible chemical modifi-
cations to DNA or nucleosomal core histones.1 Epige-
netics plays a major role in a number of biological
events, including cell differentiation, embryonic develop-
ment, inflammation, and cancer. One of the major epige-
netic activities is the acetylation of lysine side chains of
histone tails, which is carried out by a family of histone
acetyltransferases (HATs) and reversed by histone
deacetylases (HDACs).1 HDAC inhibitors have been
shown to have antiproliferative effects on tumor cells
in vitro through a number of mechanisms including cell
cycle arrest, apoptosis, and induction of terminal differ-
entiation.1–4 In vivo efficacy in xenograft tumor models
has further substantiated HDACs as valid target for
developing novel anticancer agents.5,6 Both hydroxamic
acid-based HDAC inhibitors, for example, Vorinostat
(SAHA)7,8 (1), LBH5899–12 (2), and PXD10113,14 (3),
and non-hydroxamic acid-based HDAC inhibitors, for
example, MS27515,16 (4) and FK22817 (Fig. 1), are cur-
rently in the clinic. Clinical trials to date have shown
these inhibitors to have activity against a variety of
malignancies.3,4 SAHA is the first HDAC inhibitor to
be approved by the FDA for the treatment of cutaneous
T-cell lymphoma. The non-hydroxamate inhibitor
MS275 has been examined in a phase I trial,16
LBH589 has been tested in hematologic malignancies,12
and PXD101 has been tested in an ovarian cancer
trial.14 In each case histone hyperacetylation was ob-
served in peripheral blood mononuclear cells.
The bacterial HDAC homolog HDLP18 and human
HDAC819,20 has been co-crystallized with hydroxamic
acid-based inhibitor, SAHA. Several HDAC inhibitors
have been designed and synthesized based on these crys-
tallographic data.21–24 In all of these designs the com-
mon structural motif for an HDAC inhibitor consists
of a metal binding head group (A), which interacts with
the Zn2+ ion at the bottom of the active site, a linker do-
main (B), which occupies the narrow tubular pocket,
and a cap group (C), which interacts with the residues
on the rim of the active site (5, Fig. 1).
In our present study, we have designed novel HDAC
inhibitors 6 with a hydroxamic acid as the zinc chelating
head group attached directly to a thiazole ring with a
piperazine spacer and a sulfonamide cap (Fig. 2). A
modification to the cap region leading to amides, as in
7, or N-alkyl/arylamines, as in 8, was also synthesized
(Fig. 2). We also prepared a series of sulfonamides 9
with the zinc chelating hydroxamic acid extended from
the thiazole ring via a double bond for comparison of
Keywords: HDAC; Inhibitors; Synthesis; Antiproliferative.
*
Corresponding author. Tel.: +1 510 785 7060x210; fax: +1 510 785
7061; e-mail: skumar@aretetherapeutics.com
Present address: Arete Therapeutics, 3912 Trust Way, Hayward, CA
94545, USA.
à
Present address: Gilead Sciences Inc., 333 Lakeside Drive, Foster
City, CA 94404, USA.
§
Present address: Tetraphase Pharmaceuticals Inc., 480 Arsenal
Street, Watertown, MA 02472, USA.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.050

5996
S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5995–5999
O
O
H
N
NHOH
H
N
NHOH
HN
O
O
SAHA (1)
LBH (2)
O
O
O
N
NH2
O
H
H
S
N
N
NHOH
N
H
O
PXD101 (3)
MS275 (4)
B
5
A
C
Figure 1. HDAC inhibitors in clinical trial and their structural motif.
12 which on treatment with 50% aqueous hydroxyl-
amine in the presence of sodium hydroxide resulted in
the desired hydroxamic acids 6.
O
S
O
R
O
O
N
N
S
S
Ar
N
N
NHOH
NHOH
N
The preparation of amide analogs of hydroxamic acids 7
was carried out according to Scheme 2. The intermediate
N
6 (R = SO2Ar)
7 (R = COR1)
8 (R = R2)
9
O
HN
R1
Figure 2. Thiazole based hydroxamic acid HDAC inhibitors.
N
a
N
O
S
N
O
S
N
OMe
HDAC enzyme activity with that of the 6 series. Here we
report the synthesis and biological evaluation of these
novel HDAC inhibitors.
N
11
OMe
13
b
Sulfonamide analogs of hydroxamic acids 6 were syn-
thesized from the commercially available 2-bromothiaz-
ole ester 10 (Scheme 1). Accordingly, methyl 2-
bromothiazole carboxylate 10 was treated with pipera-
zine in the presence of potassium carbonate to obtain
thiazole intermediate 11. The intermediate 11 was trea-
ted with arylsulfonyl chlorides to afford sulfonamides
O
R1
N
N
N
O
S
N
NHOH
7
R2
N
HN
R2
H
Br
N
O
S
Br
a
O
S
c
N
O
S
+
+
N
N
O
OMe
S
N
OMe
N
H
OMe
N
H
N
OMe
10
N
15
10
11
14
b
b
O O
S
O O
S
R2
Ar
N
N
Ar
N
c
N
N
O
O
S
N
S
O
S
N
N
N
12
NHOH
NHOH
OMe
6
8
Scheme 1. Reagents and conditions: (a) K2CO3, MeCN, 80 °C, 8 h
(90%); (b) arylsulfonyl chlorides, Et3N, DCM, rt, 4–12 h (87%); (c)
50% aq NH2OH, NaOH, MeOH, DCM, rt, 4–5 h (50%).
Scheme 2. Reagents and conditions: (a) alkyl/arylacid chlorides, Et3N,
DCM, rt, 4–12 h (86%); (b) 50% aq NH2OH, NaOH, MeOH, DCM,
rt, 4–5 h (50%); (c) K2CO3, MeCN, 80 °C, 8 h (92%).

S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5995–5999
5997
ester 11 was treated with either acetyl chloride, pheny-
lacetyl chloride or benzoyl chloride in the presence of
triethylamine to obtain the corresponding amide inter-
mediates 13. Amide intermediates 13 were then con-
verted to hydroxamic acids 7 by reaction with 50%
aqueous hydroxylamine in the presence of sodium
hydroxide. Synthesis of the corresponding N-alkyl/aryl
analogs 8 is also shown in Scheme 2. Accordingly,
bromothiazole 10 was reacted with the commercially
available N-alkyl or aryl piperazines 14 in the presence
of potassium carbonate to obtain esters 15, which were
then converted to hydroxamic acids 8 by treatment with
aqueous hydroxylamine and sodium hydroxide.
wards the breast cancer cell line MCF7 were determined
for hydroxamic acids 6, 7, 8, and 9. SAHA26 was used as
positive control, affording an IC50 value of 0.06 lM and
a GI50 value of 0.4 lM. The data for hydroxamic acids 6
(Table 1) clearly indicate that these novel hydroxamates
potently inhibit HDAC enzymes with the best activity
seen for compounds 6c and 6f with IC50 values of
<100 nM. The SAR data suggest that electron-donating
groups on the aryl ring afford more potent inhibitors (6c
and 6f) compared to electron-withdrawing groups (6b
Table 1. HDAC inhibition and cell proliferation data for hydroxamic
acids 6, 7, and 8a
Compound Ar/R1/R2
HDAC
MCF7
The a, b-unsaturated hydroxamic acids 9 were synthe-
sized according to Scheme 3. Commercially available
2-bromo-5-formylthiazole 16 was treated with N-Boc-
piperazine and potassium carbonate to obtain formyl
intermediate 17. Intermediate 17 was converted to the
a, b-unsaturated ester 18 via a Wittig–Horner reaction
with trimethyl 2-phosphonoacetate in the presence of
n-butyl lithium followed by Boc deprotection. The a,
b-unsaturated ester intermediate 18 was then treated
with arylsulfonyl chlorides in the presence of triethyl-
amine to obtain the sulfonamide esters 19. The acids
resulting from ester hydrolysis of 19 were coupled with
tetrahydropyranyl (THP) protected hydroxylamine in
presence of hydroxybenzotriazole and 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide to give the THP pro-
tected a, b-unsaturated hydroxamic acid intermediates
20. Removal of the THP group in 20 using hydrochloric
acid in ether afforded the desired hydroxamic acids 9.
IC50 (lM) GI50 (lM)
Sulfonamides
6a
6b
6c
6d
6e
6f
2-Naphthyl
4-Trifluoromethoxyphenyl 1.2
0.22
2
4
4-Methylphenyl
4-Biphenyl
0.09
0.2
0.35
1.8
4
4-Trifluoromethylphenyl
3,4-Dimethoxyphenyl
Phenyl
2.6
0.07
0.57
0.4
2
6g
6h
6i
10
10
3.5
4-Nitrophenyl
4-Fluorophenyl
0.15
Amides
7a
7b
Methyl
Phenyl
Benzyl
2.8
3.5
32
>100
40
20
7c
Aryl/alkyl amines
8a
8b
8c
Methyl
Benzyl
2-(Phenyl)ethyl
>100
>100
>100
>100
4
>100
The in vitro HDAC inhibitory activity in a HeLa cell
nuclear extract25 and the antiproliferative activity to-
a Average value of at least two independent experiments.
Boc
N
Br
S
N
S
a
+
HN
CHO
CHO
N
N
N
Boc
16
17
b, c
O O
S
Ar
HN
N
OMe
d
N
S
N
S
O
O
N
N
18
OMe
19
e, f
O O
S
O O
S
Ar
N
Ar
N
N
N
S
g
S
O
O
N
N
NHOTHP
20
NHOH
9
Scheme 3. Reagents and conditions: (a) K2CO3, MeCN, 80 °C, 8 h (92%); (b) trimethylphosphonoacetate, n-BuLi, THF, À50 °C, 2 h (74%); (c) 4 M
HCl/dioxane, 2 h (97%); (d) arylsulfonyl chlorides, Et3N, DCM, rt, 4–12 h (86%); (e) NaOH, MeOH, rt, 5 h (95%); (f) HN2OTHP, EDCI, HOBT,
Et3N, DCM, rt, 12 h (83%); (g) HCl, MeOH, Et2O, rt, 5 h (91%).

5998
S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5995–5999
and 6e). The sulfonamide analogs 6 show good potency
against HDAC enzymes, while the corresponding amide
analogs 7 and alkyl/aryl analogs 8 were less potent
against the HDAC enzyme and showed less antiprolifer-
ative activity against the breast tumor cell line (Table 1).
The antiproliferative activity (GI50) toward MCF7 for
sulfonamides 6 was in the range of 0.35–10 lM with
the best cellular activity seen for 6c which exhibited a
GI50 of 350 nM.
References and notes
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Having identified the sulfonamide functionality as the
optimal cap group, we explored the possibility of
improving both HDAC enzyme activity and cell po-
tency by preparing the a, b-unsaturated hydroxamic
acids 9. Such a, b-unsaturated hydroxamic acids, for
example, LAQ824,7 LBH589,7 (2), and PXD1019 (3),
have been shown to be potent HDAC inhibitors.
The results presented in Table 2 suggest that such a
modification does not, in general, result in improved
potency in this series, except for 9a. Seven compounds
out of the nine in the 6 series displayed greater po-
tency against HDAC enzyme than the corresponding
a, b-unsaturated analog in the 9 series, with differ-
ences of approximately 2- to 50-fold (e.g., 6d, HDAC
IC50 of 0.2 lM vs 9d, HDAC IC50 of 11 lM). There
was not a direct correlation between HDAC inhibitory
activity and cellular potency for all pairwise compari-
sons within the 6 and 9 series, however 6 analogs
demonstrated higher enzyme inhibitory activity and
increased antiproliferative activity, for example, 6c
(IC50 0.09 lM, GI50 0.35 lM) versus 9c (IC50
0.55 lM, GI50 2 lM). The lack of correlation in cellu-
lar potency of the 9 series versus the 6 series com-
pounds may be due to improved cellular
permeability as a result of the a, b-unsaturated
hydroxamic acid function.
In summary, we have designed and synthesized a series
of novel HDAC inhibitors having both enzymatic and
antiproliferative activity. The sulfonamide analog 6c
was found to have both enzyme and cell potency similar
to SAHA. Future efforts will involve the identification of
readily synthesized analogs of series 6, which are potent
and chemically stable HDAC inhibitors that avoid any
liability resulting from the a, b-unsaturated double bond
present in series 9.
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acids 9a
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Compound Ar
HDAC
MCF7
IC50 (lM) GI50 (lM)
9a
9b
9c
9d
9e
9f
2-Napthyl
0.05
3.2
0.7
6
4-Trifluoromethoxyphenyl
4-Methylphenyl
4-Biphenyl
0.55
2
11
15
0.16
1.5
3
4-Trifluoromethylphenyl
3,4-Dimethoxyphenyl
Phenyl
4
9g
9h
9i
0.44
1.6
3
4-Nitrophenyl
4-Fluorophenyl
5
2.5
4.7
a Average value of at least two independent experiments.

S.-K. Anandan et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5995–5999
5999
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extract, which contains a number of HDAC isozymes
and other nuclear factors, was used as the source of
HDAC activity. The final substrate concentration in the
assay mixture was 50 lM. The reaction was allowed to
proceed for 10 min at room temperature before a stop
solution was added. Test compounds were prepared as
20 mM stock solutions in DMSO (Molecular Biology
grade, Sigma–Aldrich Co., St. Louis, MO) and stored at
À70 °C. DMSO had no significant effect on the activity
of this assay at concentrations up to 5% with the final
DMSO concentration in the assays of no more than 2%.
Assays were performed in white polystyrene 96-well
half-area assay plates (Corning, Corning, NY) and
measured on a Wallac 1420 fluorescent plate reader
(Wallac Oy, Turku, Finland) with an excitation wave-
length of 355 nm, an emission wavelength of 460 nm,
and a one sec signal averaging time.
25. In vitro fluorescent histone deacetylase assay: HDAC
inhibition assays were performed using the HDAC
fluorescent activity assay kit (Biomol Research Labora-
tories, Plymouth Meeting, PA). HeLa cell nuclear
26. Mai, A.; Esposito, M.; Sbardella, G.; Massa, S. A. Org.
Prep. Proced. Int. 2001, 33, 391.