(2-Amino-phenyl)-amides of ω-substituted alkanoic acids as new histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.08.083

A variety of ω-substituted alkanoic acid (2-amino-phenyl)-amides were designed and synthesized. These compounds were shown to inhibit recombinant human histone deacetylases (HDACs) with IC₅₀ values in the low micromolar range and induce hyperacetylation of histones in whole cells. They induced expression of p21WAF1/Cip1 and caused cell-cycle arrest in human cancer cells. Compounds in this class showed efficacy in human tumor xenograft models.

Full text of DOI:10.1016/j.bmcl.2003.08.083

Bioorganic & Medicinal Chemistry Letters 14 (2004) 283–287
(2-Amino-phenyl)-amides of !-substituted alkanoic acids as new
histone deacetylase inhibitors
Arkadii Vaisburg,a,* Naomy Bernstein,a Sylvie Frechette,a Martin Allan,a
Elie Abou-Khalil,a Silvana Leit,a Oscar Moradei,a Giliane Bouchain,a James Wang,a
Soon Hyung Woo,a Marielle Fournel,b Pu T. Yan,b Marie-Claude Trachy-Bourget,b
Ann Kalita,b Carole Beaulieu,b Zuomei Li,b A. Robert MacLeod,b Jeffrey M. Bestermanb
and Daniel Delormea,*,y
aDepartment of Medicinal Chemistry, MethylGene Inc., 7220 Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
bDepartment of Molecular Biology, MethylGene Inc., 7220 Frederick-Banting, Montreal, Quebec, Canada H4S 2A1
Received 2 June 2003; accepted 21 August 2003
Abstract—A variety of o-substituted alkanoic acid (2-amino-phenyl)-amides were designed and synthesized. These compounds
were shown to inhibit recombinant human histone deacetylases (HDACs) with IC50 values in the low micromolar range and induce
hyperacetylation of histones in whole cells. They induced expression of p21WAF1/Cip1 and caused cell-cycle arrest in human
cancer cells. Compounds in this class showed efficacy in human tumor xenograft models.
# 2003 Elsevier Ltd. All rights reserved.
Identification of potent HDAC inhibitors possessing
adequate ‘drug-like’ properties represents a considerable
effort in the development of therapeutics for the treat-
ment of cancers. In eukaryotic cells, histone acetylation/
deacetylation is co-regulated by enzymes called histone
acetyltransferases (HATs) and histone deacetylases
(HDACs).1 Acetylation of histones is involved in many
cellular functions including chromatin remodeling and
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 malig-
nant diseases.3 Small molecules hydroxamic acids,
including the natural product trichostatin A (1, TSA)4
or its analogues,5 suberoylanilide hydroxamic acid (2,
SAHA),6 scriptaid (3)7 or its analogues,8 oxamflatin (4)9
or the 2-aminoanilide MS-275 (5),10 are potent HDAC
inhibitors (Fig. 1).
hydroxycinnamides 6 and arylsulfonamido-2-amino-
phenyl cinnamides 7.11 The next class of compounds
was SAHA-TSA-like straight-chain hydroxamates 8.12
Here we describe the synthesis and biological activity of
novel 2-aminophenylamides of o-substituted alkanoic
acids 9–10, structurally relevant to compounds 8 and
having the same optimized chain length (Fig. 2). As
HDACs are viewed as an important new family of can-
cer targets, efforts to identify non-hydroxamate small
molecule HDAC inhibitors are underway in many
research groups working in this area. For instance, a
In our efforts to identify novel HDAC inhibitors we
initially designed and synthesized arylsulfonamido-N-
* Corresponding authors. Tel.: +1-514-337-3333; fax: +1-514-337-
0550; e-mail: vaisburga@methylgene.com
y
Present address: PhageTech Inc., 7170 Frederick-Banting, 2nd Floor,
Montreal, Quebec H4S 2A1, Canada.
Figure 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.083

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A. Vaisburg et al. / Bioorg. Med. Chem. Lett. 14 (2004) 283–287
Scheme 1. Reagents and conditions: (a) Zn/BrCH2CH2Br/Me3SiCl;
(b) LiCl/CuCN/THF; (c) p-BrC6H4COCl/THF, 80% three steps; (d)
NaOH/H2O/THF/MeOH, 94%; (e) HOBt/EDC/DMF then o-phenyl-
ene diamine, 30%.
coupling chemistry (HOBt/EDC) to afford the final
product 12b in moderate yield (Scheme 1).
Figure 2.
recent publication from Abbott Laboratories13
describes the synthesis and biological activity of a series
of long-chain trifluoromethyl ketones.
Compounds 12a and 12c–g (see Table 1) were prepared
similarly to the compound 12b, using the corresponding
arylcarbonyl chlorides as intermediates. Two pyridine-
based anilides (Scheme 2) were prepared from bromo-
pyridines. Starting from 3-bromopyridine (13), the cor-
responding boronic acid was generated in situ and then
reacted with ketoester 11 to form ketoester 14. Saponi-
fication followed by HOBt/EDC coupling produced
anilide 12h. Its isomer, compound 12i, was synthesized
from 2,5-dibromopyridine (15) which was converted to
the trimethyltin intermediate and subsequently coupled
to the monomethyl ester of suberoyl chloride under
Stille conditions. The bromopyridyl ketoester 16 under-
went Suzuki coupling with phenylboronic acid to yield
the intermediate 17, which after saponification and
treatment with o-phenylenediamine afforded compound
12i.
The first series of target compounds, phenyl, biphenyl
and naphthyl anilides 12a–g (Table 1) was synthesized
starting from ethyl 7-iodoheptanoate as outlined in
Scheme 1 (exemplified by anilide 12b).12 Formation of
the copper-zinc organometallic intermediate, followed
by reaction with p-bromobenzoyl chloride yielded com-
pound 11. This aryl ketoester was saponified and then
coupled to o-phenylenediamine using standard peptide
Table 1. 8-Oxo-8-aryl-octanoic acid (2-amino-phenyl)-amides
A series of heterocyclic derivatives 20a–g (Table 2) were
prepared via condensation reactions (Scheme 3).
o-Phthalaldehyde (18) was readily converted to oxo-
No.
R
HDAC-1
IC50 (mM)
MTT HCT116
IC50 (mM)
H4-Ac
EC50 (mM)
12a
9
>25
20
21
9
25
15
5
12b
12c
12d
12e
12f
12g
12h
12i
6
5
2
25
25
10
3
1
12
7
2
4
4
Scheme 2. Reagents and conditions: (a) n-BuLi, Et2O, hexane then
B(OMe)3; (b) 11, Pd(Ph3)4, K2CO3, toluene, H2O, 25%; (c) NaOH,
H2O, THF, MeOH, 75%; (d) HOBt, EDC, DMF then o-phenylene
diamine, 6%; (e) n-BuLi then Me3SnCl; (f) ClCO(CH2)6CO2Me,
PdCl2(PPh3)2, 39% two steps; (g) PhB(OH)2, Pd(Ph3)4, K2CO3, tolu-
ene, EtOH, 88%; (h) NaOH, H2O, THF, MeOH, 90%; (i) HOBt,
EDC, DMF then o-phenylene diamine, 33%.
10
3
19
5
20
5

A. Vaisburg et al. / Bioorg. Med. Chem. Lett. 14 (2004) 283–287
285
isoindole 19 using known procedure14 and then to the
final product 20a. Compounds 20b–e were prepared
starting from the appropriate phthalic anhydrides
reacting with 6-aminohexanoic acid, followed by pep-
tide coupling with o-phenylenediamine (Scheme 3). In
the case of 20e, where the desired anhydride was
not commercially available, it was prepared from
4-hydroxyphthalic acid (21) simply by heating under
vacuum. Mannich reaction was employed to convert
the hydroxy-substituted phthalimide 23 to piper-
idinylmethyl-intermediate 24 which led to compound
20f.
28b. Reaction of isatoic anhydride (25) with 6-amino-
hexanoic acid followed by consecutive treatment of the
resultant reaction mixture with methyl chloroformate
and then a base afforded quinazolinedione 30.16 This
material upon alkylation with methyl iodide followed by
reaction with an alkali led to 29. Both acids 30 and 29
were coupled in the usual manner to form compounds
28c and 28d.
Naphthalimides 28e–g (Table 3) were derived from the
corresponding naphthalic anhydrides using chemistry
depicted in the Scheme 3.
Compounds 28e–28g (see Table 3) were prepared simi-
larly to the compound 20e, from the appropriately sub-
stituted phthalic or naphthalic anhydrides. Compounds
20g, 28h, and 28i (see Tables 2 and 3) were prepared
similarly to compound 20f.
All compounds were initially screened for the ability to
inhibit recombinant human HDAC-1.17 We chose this
isozyme since it is widely implicated in transcriptional
repression and chromatin remodeling. The first com-
pound in this series 12a showed modest activity against
HDAC-1 with the IC50 of 9 mM (Table 1). Introduction
of substituents on the phenyl ring (compounds 12b and
12c) slightly improved their activity while increasing the
size of the entire aryl fragment, compounds 12d–12f,
resulted in a 5-fold gain in potency (IC50 1–2 mM, Table
1). Neither a moderate increase of the size of the aryl
substituent nor introduction of a basic site (compounds
12g and 12h) proved to be beneficial. However, the iso-
mer of 12h, compound 12i, showed potency similar to
the one of compounds 12e and 12f (Table 1).
Scheme 4 outlines the synthesis of several quinazoli-
nones 28a, 28c, 28d, and benzotriazinone 28b, all stem-
ming from the same starting material. Reaction of
isatoic anhydride (25) with 6-aminohexanoic acid fol-
lowed by cyclization in the presence of either formic
acid or sodium nitrite/HCl resulted in quinazolinone
(26: X=CH) or benzotriazinone (27: X=N), respec-
tively.15 Each was then coupled to yield anilides 28a and
Table 2. 6-Heteroaryl-hexanoic acid (2-amino-phenyl)-amides
To explore more comprehensively the SAR of this series
we hypothesized that restrictions in the flexibility of the
chain between the carbonyl groups may improve
potency. The first two compounds of this type were the
bi-cyclic compounds of the isoindole series 20a and 20b,
having HDAC-1 potency of 4 and 2 mM, respectively
No.
R1
HDAC-1
IC50 (mM)
MTT HCT116
IC50 (mM)
H4-Ac
EC50 (mM)
20a
4
2
15
24
99
5
20b
20c
20d
20e
5
3
3
>47
6
>25
3
11
10
20f
3
4
31
12
—
—
Scheme 3. Reagents and conditions: (a) H2N(CH2)5COOH, AcOH,
reflux 97%; (b) HOBt, EDC, DMF then o-phenylene diamine 27%;
(c) Á, 220 ꢀC, 2 h; (d) H2N(CH2)5COOH, AcOH, reflux 98%;
(e) HOBt, EDC, DMF then o-phenylene diamine 18%; (f) paraform,
piperidine, dioxane, DMF 37%; (g) HOBt, EDC, DMF then o-phen-
ylene diamine 44%.
20g

286
A. Vaisburg et al. / Bioorg. Med. Chem. Lett. 14 (2004) 283–287
Table 3. 6-Heteroaryl-hexanoic acid (2-amino-phenyl)-amides
No.
R2
HDAC-1
IC50 (mM)
MTT HCT116
IC50 (mM)
H4-Ac
EC50 (mM)
28a
12
8
10
10
5
5
10
10
—
28b
28c
28d
4
Scheme 4. Reagents and conditions: (a) H2N(CH2)5COOH, Et3N,
H2O then HCOOH, reflux; (b) H2N(CH2)5COOH, Et3N then NaNO2,
HCl; (c) H2N(CH2)5COOH, Et3N, MeOCOCl then NaOH 45%;
(d) HOBt, EDC, DMF then o-phenylene diamine; (e) MeI, K2CO3
then NaOH 90%.
4
5
28e
28f
28g
3
1
1
2
3
10
5
To confirm the ability of the amino anilides to inhibit
HDAC in whole cells, all compounds were evaluated for
the ability to induce histone acetylation in T24 human
bladder cancer cells. The anilides (12f, 12i, 28e, 28g, and
28i) caused dose-dependent histone hyperacetylation in
these cells (Tables 1–3, Fig. 3). Similarly to other known
HDAC inhibitors the compounds 12f, 12i, 28e, 28g and
28i also induced expression of p21WAF1/Cip1 protein
in T24 human cancer cell lines (Fig. 4), while com-
pounds 12f and 28g caused G2/M cell cycle arrest in
HCT116 (Fig. 5).
6
0.9
28h
28i
2
1
1
HDAC small molecule inhibitors with attractive pro-
files from in vitro screening (12f, 12i, 28e, 28g, and 28i)
were evaluated in vivo in several different human
tumor xenograft models: A549 (non-small cell lung),
PANC-1 (pancreatic), DU145 (prostate) and HCT116,
Colo205 and SW48 (colon). Compounds were admi-
nistered daily by either intraperitoneal (ip) injection or
orally (po). Compounds 12f, 12i, 28e, 28g, and 28i
0.5
1
1
2
1
MS-275
0.5
(Table 2). Introduction of a variety of substituents on
the phenyl ring (compounds 20d–g) or replacing phenyl
with pyridine (compound 20c) did not improve the
potency (Table 2), prompting us to prepare compounds
of quinazolinone type 28a–d and later benzoisoquinoli-
nediones 28e–i (Table 3). The last four turned out to be
quite potent, in fact, as potent as MS-275 (5) which is
currently undergoing phase I clinical trials.18
We evaluated the in vitro antiproliferative activities of
the compounds using the 3-[4,5-dimethylthiazol-2-yl-
2,5-diphenyltetrazolium] bromide (MTT) assay against
various human cancer cell lines, in particular HCT116
(human colon cancer, Tables 1–3). The compounds
showed a range of antiproliferative activities from 1 to
50 mM.
Figure 3. Human bladder carcinoma T24 cells were treated with
compounds at 0, 1, 5, and 25 mM for 16 h. Cells were harvested
and histones were acid-extracted, analyzed by SDS-PAGE and
immunoblotting with antibodies specific for acetylated H4 histones.
Histones were stained by Coomassie Blue to reveal their amount
loaded on the gels.

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287
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showed measurable antitumor activity in vivo. Com-
pounds 12f and 28g displayed the greatest antitumor
activity with % TGIs (tumor growth inhibitions rela-
tive to vehicle controls) of 53 and 28%, respectively,
when dosed at 40 mg/kg per day for 3 weeks. This
activity was obtained without any associated body
weight loss as a measure of gross toxicity. However the
activity was lower than that of MS-275 (5) which was
used as a positive control in these experiments. These
results may be attributed to relatively short half-life
and poor bioavailability of the listed entities (data not
shown).
In summary, the (2-amino-phenyl)-amides of o-sub-
stituted alkanoic acids were shown to inhibit recombi-
nant human HDACs with IC50 values in the low
micromolar range, induce hyperacetylation of histones,
increase expression of p21WAF1/Cip1 and cause cell-
cycle arrest in human cancer cells in a dose dependent
manner. Although the title compounds are not yet com-
pletely optimized, the results indicate a significant step
towards the development of small molecule HDAC inhi-
bitors with acceptable pharmaceutical characteristics and
potent antitumor activity.
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