Design and synthesis of a novel class of histone deacetylase inhibitors.

Article abstract of DOI:10.1016/S0960-894X(01)00552-2

Histone deacetylase inhibitors (HDACs) have emerged as a novel class of antiproliferative agents. Utilizing structure-based design, the synthesis of a series of sulfonamide hydroxamic acids is described. Further optimization of this series by substitution of the terminal aromatic ring yielded HDAC inhibitors with good in vitro and in vivo activities.

Full text of DOI:10.1016/S0960-894X(01)00552-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2847–2850
Design and Synthesis of a Novel Class of Histone
Deacetylase Inhibitors
Rico Lavoie,a,y Giliane Bouchain,a Sylvie Frechette,a Soon Hyung Woo,a
Elie Abou Khalil,a Silvana Leit,a Marielle Fournel,b Pu T. Yan,b
Marie-Claude Trachy-Bourget,b Carole Beaulieu,b Zuomei Li,b
Jeffrey Bestermanb and Daniel Delormea,*
a
´
´
Department of Medicinal Chemistry, MethylGene Inc., 7220 Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
b
´ ´
Department of Biology, MethylGene Inc., 7220 Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
Received 6 July 2001; accepted 3 August 2001
Abstract—Histone deacetylase inhibitors (HDACs) have emerged as a novel class of antiproliferative agents. Utilizing structure-
based design, the synthesis of a series of sulfonamide hydroxamic acids is described. Further optimization of this series by sub-
stitution of the terminal aromatic ring yielded HDAC inhibitors with good in vitro and in vivo activities. # 2001 Elsevier Science
Ltd. All rights reserved.
The identification of potent HDAC inhibitors repre-
sents a considerable effort in the development of ther-
apeutics for treatment of cancers. In eukaryotic cells,
histone acetylation/deacetylation, which is co-regulated
by enzymes called histone acetyltransferases (HATs)
and histone deacetylases (HDACs), is essential for
chromatin remodeling and the functional regulation of
gene transcription.1 HDACs modulate the deacetylation
of e-amino groups of lysines located near the N-termini
of core histone proteins.2 Deregulation of HDAC
activity is implicated in malignant diseases.3 Small
molecules having a hydroxamic acid functional group
are highly potent HDAC inhibitors, such as the natural
product trichostatin A (TSA)4 (1) or analogues,5 sub-
eroylanilide hydroxamic acid (SAHA)6 (2), and the ene-
yne oxamflatin (3).7
For the first set of structural modifications, we have
prepared sulfonamides having a hydroxamate as the
zinc chelating moiety. Based on previous work, it has
been shown that this function is required for the HDAC
activity of TSA and TSA-like hydroxamate analogues.
A synthetic route for preparing sulfonamides having
different chain length is outlined in Scheme 1. Ester-
ification of carboxylic acid 5, followed by the treatment
with phenylsulfonyl chloride in the presence of triethyl-
amine and ester saponification afforded the compound
6. The conversion of acid 6 to the hydroxamic acid 7
was accomplished by coupling with a THP-protected
hydroxylamine and the subsequent deprotection under
acidic conditions.
In the course of our studies to design and develop inhi-
bitors of HDACs, we identified promising lead sub-
stances having the general structure 4.8
In this paper, we describe our preliminary results on the
synthesis of a series of sulfonamides and their biological
evaluation as HDAC inhibitors.
A five-step synthesis of compounds 11 and 12 is depicted
in Scheme 2. Iodoaniline 8 was treated with an excess of
aryl sulfonyl chloride in the presence of triethylamine,
*Corresponding author. Tel.: +1-514-337-3333; fax: +1-514-337-
0550; e-mail: delormed@methylgene.com
yPresent address: Bayer Co. 400 Morgan Lane West Haven CT, USA.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00552-2

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R. Lavoie et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2847–2850
the treatment with these compounds induced histone
acetylation, the human bladder carcinoma T24 cells
were chosen.
For HDAC inhibitory activity, the optimal chain length
between the aryl and the hydroxamate was found to be
two carbons which was comparable to the unsaturated
chain (12a, 11a, IC50=0.1, 0.2 mM, respectively, in
HDAC-1). The benzamide (7a), the methylene (7b), and
the propyl (7c) decreased the potency by 10-fold.
Scheme 1. Conditions: (a) HCl, MeOH; (b) PhSO2Cl, Et3N, DCM
(96–99%); (c) LiOH, H2O/THF; (d) NH2OTHP, EDC, HOBt, DMF,
50 ꢀC (50–60%); (e) CSA, MeOH.
By substituting adjacent to the amide bond, 11b was
found to be 10-fold less potent than the corresponding
unsubstituted compound (11a). A dramatic decrease in
potency (170-fold) was observed when the saturated
analogue (12b) was substituted with a methyl group.
The more hindered isopropyl was also detrimental for
HDAC inhibition (17, inactive). These results suggest
that steric hindrance is not acceptable near the zinc
binding site and is in agreement with the proposed nar-
rowness of the binding site based on the co-crystal
structure of the bacterial HDAC-1 enzyme with the
hydroxamic acid TSA.10
Scheme 2. Conditions: (a) ArSO2Cl, Et3N, DCM (96–99%); (b)
NaOMe, MeOH; (c) Pd2(dba)3, P(o-tolyl)3, CH2¼CRCOOH, Et3N,
DMF, 100 ꢀC (97–99%); (d) NH2OTHP, EDC, HOBt, DMF, 50 ꢀC
(55–65%); (e) CSA, MeOH; (f) H2, Pd/C.
followed by the treatment with sodium methoxide to
provide the aryl sulfonamide (9). Compound 10 was
prepared using a Heck coupling reaction of iodide 9
with acrylic acid in the presence of Pd2(dba)3 and tri(o-
tolyl) phosphine. Subsequently, acid 10 was converted
to the hydroxamic acid 11. Hydrogenation of alkene 10
yielded the acid which was converted to the hydroxamic
acid (12).
N-Methylation of the sulfonamide (18a) resulted in a
decrease in inhibitory activity. None of the parent car-
boxylic acid analogues was active.
Although compounds 11a and 12a have comparable
biological activity, for simplicity, only the unsaturated
compound was explored for further chemistry. The sul-
The a-substituted hydroxamic acid (17) was synthetized
as shown in Scheme 3. The condensation of 4-nitro-
bromo-benzyl 13 and ethyl isobutyrate 14 in the pre-
sence of LDA afforded ester 15. Reduction of the nitro
group, followed by treatment with phenylsulfonyl chlo-
ride in the presence of triethylamine afforded compound
16. Saponification of 16 under alkaline conditions
afforded the acid which was coupled with hydrox-
ylamine to give the target hydroxamic acid (17).
Table 1. HDAC-1 inhibition, cell proliferation quantification, and
histone acetylation for 7a–18
Compd
YR
HDAC-1
MTT
H4-Ac
IC50 (mM)a EC50 (mM)b EC50 (mM)c
(ꢁ)TSA
7a
7b
12a
7c
0.005
0.9
1
0.1
1
1
2
22
2
1
5
>25
5
—
CH2
(CH2)2
(CH2)3
H
H
H
H
8
>25
11a
11b
H
H
0.2
2
3
5
15
>25
Scheme 3. Conditions: (a) LDA, THF; (b) H2, Pd/C; (c) (i) PhSO2Cl,
Et3N, DCM; (ii) MeONa, MeOH (80%); (d) LiOH, H2O/THF; (e)
.
NH2OH HCl, Et3N, EDC, HOBt, rt, 12 h (88%).
12b
17
H
H
17
>50
30
>25
>25
>25
The SAR of representative chain lengths for the tem-
plate 18 is presented in Table 1.
>25
In vitro activity was measured through the inhibition of
partially purified recombinant human HDAC-1.9 The
antiproliferative activity of these compounds was ana-
lyzed by MTT assays (spectrophotometric quantifica-
tion of viable cells by cleavage of tetrazolium salt). HCT
116 cells were chosen for MTT assays because they have
a high level of HDAC expression. To discover whether
18a
CH3
0.6
4
aValues are means of three experiments.
bIn vitro antiproliferative activity against human colon cancer HCT
116 cells. Values are means of two experiments.
cConcentration of inhibitor for acetylation of histone-4 in human T24
cancer cells.

R. Lavoie et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2847–2850
2849
fonamides 11 were prepared with various substitution
on the left phenyl ring (Table 2).
out to be less active than the 4-chloro analogue. This
might be ascribed to either an unfavorable steric effect
of meta- and ortho- substitution or to exceeding the
optimum lipophilic value of the substituents. This result
seems to be confirmed by the m-trifluoromethyl analo-
gue (11i). A more electron withdrawing group such as a
nitro or a sulfonamide in the para position seem to be
deleterious for potency since 11f and 11g are 10-fold less
active than 11c. Whereas the para-methyl (11j), meta-
methyl (11k) and para-tbutyl (11l) analogues display
almost the same potency as the parent compound, the
activity seems to depend on the steric effect since the tri-
isopropyl substituted analogue (11m) is less potent. The
para-methoxy (11n) and the 3,4-dimethoxy (11o) analo-
gues are equipotent to 11c in HDAC-1, but show more
activity in MTT. Compounds 11n and 11o have sig-
nificant antiproliferative activity and can induce core
histone acetylation at doses as low as 1–5 mM. The
ability of these compounds to induce histone acetylation
correlates well with their ability to inhibit HDAC-1 in
vitro. Compound 11o was selected for further in vivo
antitumor efficacy studies and was injected intraper-
itoneally daily into nude mice bearing subcutaneous
non-small cell lung carcinoma A549 tumors, for 21
days, at doses of 50, 40, and 20 mg/kg. Tumor growth of
the treated mice was inhibited by 57, 50, and 41%,
respectively, compared to that of mice in the control
group. Gross toxicity, as measured by loss of body
weight, was not observed.
The first analogue synthesized was the p-chlorophenyl
(11c), which showed a greater potency for HDAC inhi-
bition and MTT than the parent compound 11a. The
potency of the 3,4-dichloro (11d) and 2,4-dichloro-
phenyl (11e) did not cause the desired effect and turned
Table 2. HDAC-1 inhibition, cell proliferation quantification, and
histone acetylation for 11c–11o
Compd
Ar
HDAC-1
MTT
H4-Ac
IC50 (mM)a EC50 (mM)b EC50 (mM)c
11c
0.075
1
1.5
5
11d
11e
11f
11g
11h
11i
0.1
0.3
0.7
0.8
0.6
0.1
0.3
0.1
0.1
2
4
>25
6
20
>25
2
The three-dimensional (3_D) structural model of human
histone deacetylase class I (HDAC1, residues 9–329)
was obtained by homology modeling using the COM-
>42
˚
POSER program as part of SYBYL 6.6. The 1.8 A-
resolution crystal structure of A. aeolicus histone dea-
cetylase-like protein (HDLP),9 which shares 35.2%
sequence identity with human HDAC1, was used as
template.
0.9
1
22
In order to model the HDAC1–Zn–TSA complex, the
TSA molecule was manually positioned into the binding
site of the HDAC1–Zn–water model after removal of
the catalytic water molecule. The TSA-like inhibitor 11o
was positioned into the HDAC1 binding site with the
hydroxamic acid anchor moiety as in the HDAC1–Zn–
TSA complex, and the rotatable bonds of the inhibitor
were conformationally sampled. The generated compu-
tational model in the HDLP catalytic core of TSA (Fig.
1) and compound 11o (Fig. 2) show hydrogen bonding
with key amino acids.
11j
0.7
0.3
0.8
10
11k
11l
12
5
The hydroxamic acid function of TSA is located in a
polar pocket formed by several residues capable of
forming hydrogen bonds (Tyr303, His140, and His141).
Compound 11o seems to form only one hydrogen bond
interaction (His140) in the binding pocket. However, it
seems that His178 interacts with the methoxy group of
the aromatic ring located outside of the binding pocket.
11m
0.6
4
>25
11n
11o
0.06
0.09
0.4
0.2
5
1
While substitution on the sulfonamide or on the double
bond a of the hydroxamic acid function was insufficient
to confer measurable activity, the substitution on the
aryl group provided an increase in potency. The results
a,b,cSee footnotes in Table 1.

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R. Lavoie et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2847–2850
gress, including new zinc chelating moieties and further
changes to the aromatic groups.
Acknowledgements
The authors thank M. F. Robert, I. Dupont, C. Bonfils
for cloning and expression of the HDAC enzyme. We
also thank Dr. T. Sulea and Dr. E. Purisima for mole-
cular modeling studies done at the Biotechnology
Research Institute.
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