Substituted N-(2-aminophenyl)-benzamides, (E)-N-(2-aminophenyl)-acrylamides and their analogues: Novel classes of histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.05.005

Inhibition of histone deacetylases (HDACs) is emerging as a new strategy in human cancer therapy. Novel 2-aminophenyl benzamides and acrylamides, that can inhibit human HDAC enzymes and induce hyperacetylation of histones in human cancer cells, have been

Full text of DOI:10.1016/j.bmcl.2006.05.005

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4048–4052
Substituted N-(2-aminophenyl)-benzamides,
(E)-N-(2-aminophenyl)-acrylamides and their analogues:
Novel classes of histone deacetylase inhibitors
a
a
a
a
Oscar Moradei,a, Silvana Leit, Nancy Zhou, Sylvie Frechette, Isabelle Paquin,
*
´
a
a
a
a,
´
´ ´
Stephane Raeppel, Frederic Gaudette, Giliane Bouchain, Soon H. Woo,
Arkadii Vaisburg,a Marielle Fournel,b Ann Kalita,b Aihua Lu,b
Marie-Claude Trachy-Bourget,b Pu T. Yan,b Jianhong Liu,b Zuomei Li,b
Jubrail Rahil,b A. Robert MacLeod,b Jeffrey M. Bestermanb and Daniel Delormea,à
aMethylGene Inc., Department of Medicinal Chemistry, 7220 Frederick-Banting, Montre´al, QC, Canada H4S 2A1
bMethylGene Inc., Department of Biology, 7220 Frederick-Banting, Montre´al, QC, Canada H4S 2A1
Received 16 March 2006; revised 29 April 2006; accepted 1 May 2006
Available online 18 May 2006
Abstract—Inhibition of histone deacetylases (HDACs) is emerging as a new strategy in human cancer therapy. Novel 2-aminophe-
nyl benzamides and acrylamides, that can inhibit human HDAC enzymes and induce hyperacetylation of histones in human cancer
cells, have been designed and synthesized. These compounds selectively inhibit proliferation and cause cell cycle arrest in various
human cancer cells but not in normal cells. The growth inhibition of 2-aminophenyl benzamides and acrylamides against human
cancer cells in vitro is reversible and is dependent on the induction of histone acetylation. Compounds of this class can significantly
reduce tumor growth in human tumor xenograft models.
Ó 2006 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) and histone acetyltrans-
O
O
O
5
H
N
ferases (HATs) are enzymes that regulate transcription
in eukaryotic cells by selectively deacetylating or acetylat-
ing the e-amino group of lysine residues located in the
NH2-terminal tails of core histones.1 Correlations be-
tween increased levels of histone acetylation and trans-
criptionally active genes have long been recognized, and
conversely, hypoacetylated nucleosomal histones are en-
riched in chromatin characterized by a virtual absence
of gene expression.2 The anionic phosphate backbone of
DNA strongly interacts with the cationic lysine residues
of histone proteins, resulting in a condensed chromatin
structure; lysine acetylation attenuates these ionic interac-
tions, relieving the highly condensed structure of
NHOH
NHOH
O
N
TSA (1)
SAHA (2)
O
O
N
H
NH2
H
N
N
MS-275 (3)
O
O
4 R=OH
5 R=
NH
O
O
S
R
N
H2N
X
H
O
Keywords: Histone deacetylase inhibitors; 2-Aminophenyl-benzamides;
2-Aminophenyl-acrylamides; HDAC; HDACI.
NH
H
N
*
Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337
0550; e-mail: moradeio@methylgene.com
n
NH2
Z
X
Present address: Celera Genomics, 180 Kimball Way, San Francisco,
CA 94080, USA.
6 Z= CH, N
n = 0, 1
à
Present address: Neurochem Inc., 275 Armand-Frappier Blvd, Laval,
Quebec, Canada H7V 4A7.
Figure 1.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.005

O. Moradei et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4048–4052
4049
chromatin and facilitating the accessibility of transcrip-
tion factors to DNA. HDACs and HATs influence many
cellular processes such as cell cycle progression and differ-
entiation. The deregulation of HDAC activity has also
been linked to carcinogenesis. Consequently, the modula-
tion of HDAC activity could be a potential therapeutic
strategy in treating human cancer.3
Small molecules containing functional groups such as a
hydroxamic acid or aminoanilide have been reported to
exhibit HDAC inhibitory activity. The natural product
trichostatin A (TSA)4 (1) and its analogues,5 synthetic
compounds such as suberoylanilide hydroxamic acid
(SAHA)6 (2), and MS-275 (3) are examples (Fig. 1).7
Some of these compounds have been shown to have po-
tent in vivo antitumor effects in tumor-bearing animals
and a few of them are currently in clinical trials. In some
cases, however, their potential for clinical drug develop-
ment is limited by lack of selectivity, cytotoxicity, low
solubility, and/or low stability.8
Table 1. SAR of some pyridin-3-yl-acrylamides
O
N
H
NH2
X
N
Compound
X
IC50 (lM)
HDAC1 MTT HCT116
7
8
9
PhCH2NH
Ph(CH2)3NH
PhNH(CH2)2NH
3
3
2
1
2
5
In the course of searching for novel HDAC inhibitors
with potency and good safety profiles, we initially de-
signed hydroxamic acid-based sulfonamides (4).9 Fur-
ther development of these HDAC inhibitors led to the
synthesis of the corresponding aniline-based sulfona-
mides (5) which showed improved pharmaceutical prop-
erties.10 More importantly, our novel arylamine anilides
(6) resulted in significant improvement in antitumor
activities both in vitro and in vivo. The structure–activ-
ity relationships (SAR), the antiproliferative activity,
and the in vivo efficacy will be discussed.
N
10
11
12
13
14
4
4
5
2
2
7
3
2
4
3
H
N
N
H
N
N
H
F
N
H
The first series of compounds, N-(2-aminophenyl)-3-
(pyridin-3-yl)acrylamides 7–16 (Table 1), were synthe-
sized starting from 2,5-dibromopyridine 17 as described
in Scheme 1 (exemplified by compounds 14 and 16).11
F3CO
N
H
MeO
MeO
N
H
Compound 18 was obtained by nucleophilic substitution
of bromine in 17 with 4-methoxybenzylamine. The for-
mation of 5-bromopicolinaldehyde 19, using n-butyl
lithium and DMF followed by a reductive amination
with 3,4,5-trimethoxyaniline, gave compound 20. Heck
couplings generated the intermediates 21 and 22 starting
from 18 and 20, respectively. These tert-butyl esters were
cleaved with TFA and then coupled to 1,2-phenylenedi-
15
16
4
3
2
3
MeO
OMe
OMe
H
MeO
MeO
N
Br
Br
tert-butyl acrylate
RNH2, 120˚C
21: R = 4-Methoxyphenyl, X = CH2, Y = NH
R
Pd2(dba)3, POT
DMF, DIPEA;120˚C
N
H
N
Br
N
17
18: R = 4-Methoxybenzyl
CO2tBu
BuLi, PhMe
-78˚C
then DMF
X
R
Y
N
Br
H
Br
tert-butyl acrylate
3,4,5-Trimethoxyaniline
MeO
N
N
22: R = 3,4,5-Trimethoxyphenyl, X = NH, Y = CH2
O
Pd2(dba)3, POT
DMF, DIPEA;120˚C
Bu2SnCl2, PhSiH3, THF; rt
N
19
MeO
TFA, DCM; rt
OMe
20
O
CO2H
1,2-Phenylenediamine
BOP, Et3N, MeCN; rt
N
X
H
R
Y
N
X
NH2
R
Y
N
14: R = 4-Methoxyphenyl, X = CH2, Y = NH
23: R = 4-Methoxyphenyl, X = CH2, Y = NH
16: R = 3,4,5-Trimethoxyphenyl, X = NH, Y = CH2
24: R = 3,4,5-Trimethoxyphenyl, X = NH, Y = CH2
Scheme 1.

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O. Moradei et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4048–4052
Table 3. SAR of some 2-aminophenyl-benzamides and 2-aminophe-
nyl-nicotinamides
amine using BOP as a coupling agent to afford final
products 14 and 16.
O
N
The corresponding (E)-N-(2-aminophenyl)cinnamides
25–35 (Table 2) were also synthesized using the chemis-
try described in Scheme 1, but starting with 4-bromoan-
iline or 4-bromobenzaldehyde instead, and introducing
the benzylamine bond by reductive amination.
H
NH2
X
Y
Compound
X
Y
IC50 (lM)
HDAC1 MTT
The analogous series of N-(2-aminophenyl)-benzamides
and N-(2-aminophenyl)-nicotinamides 36–44 (Table 3)
were also synthesized. Nicotinamides 36–38 were pre-
pared in three steps from 6-chloronicotinic acid starting
HCT116
N
H
36
37
N
N
4
3
0.5
MeO
H
N
0.8
3
Table 2. SAR of some 2-aminophenyl cinnamides
MeO
MeO
O
N
N
H
38
N
5
H
MeO
NH2
X
OMe
N
H
39
40
41
42
43
44
CH
CH
CH
2
4
3
1
Compound
X
IC50 (lM)
MeO
MeO
HDAC1 MTT HCT116
N
H
N
0.6
2
25
26
4
4
37
12
H
MeO
MeO
MeO
H
N
N
H
MeO
MeO
OMe
H
N
CH 0.6
2
27
28
29
PhNHCH2
17
17
22
11
10
—
MeO
MeO
H
N
H
N
CH
CH
2
3
0.4
2
MeO
MeO
H
N
OMe
H
N
MeO
OMe
OMe
with amide synthesis followed by nucleophilic substitu-
tion of the chloride and then deprotection of the
N-Boc group, (Scheme 2, exemplified by compound
36). The N-(2-aminophenyl)-benzamides 39–44 were
synthesized as illustrated in Scheme 3 (exemplified by
compounds 41 and 43). N-(2-Aminophenyl)-4-(benzyla-
mino) benzamide 41 and N-(2-aminophenyl)-4-
((phenylamino)methyl) benzamide 43 were obtained
according to Scheme 3; starting, respectively, with
4-aminobenzoic acid 48 and 4-formylbenzoic acid 49.
H
N
MeO
30
31
32
2
2
3
5
2
5
MeO
MeO
H
N
MeO
OMe
MeO
MeO
N
All compounds were initially screened for the ability to
inhibit recombinant human HDAC-1. This isozyme
was chosen since it is widely implicated in both tran-
scriptional repression and chromatin remodeling.12
The evaluation of the in vitro antiproliferative activities
of the synthesized compounds was performed using
3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bro-
mide (MTT) assay against various human cancer cell
lines, in particular, HCT116 human colon cancer cell
line. Compound 7, in the first series (Table 1), showed
OMe
N
H
33
34
10
5
2
N
N
H
50
F3CO
O2N
N
35
4
15
H

O. Moradei et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4048–4052
4051
MS-275
Cpd 31
O
NH2
(-)
25
5
1
0.2
25
5
1
0.2
(μM)
H4-Ac
H4
CO2H
NH
NHBoc
NHBoc
Cl
N
Cl
N
BOP, TEA, MeCN; rt
45
46
MS-275
Cpd 43
ArCH2NH2
K2CO3, DMF; rt
(-)
25
5
1
0.2
25
5
1
0.2
(μM)
H4-Ac
H4
O
O
TFA, DCM; rt
NH2
NH
NH
NHBoc
Ar
N
N
Ar
N
H
N
H
Figure 2. 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 immuno-
blotted with antibodies specific for acetylated H4 histones. Histones
were stained by Coomassie blue to reveal their amount loaded on the
blots.
47: Ar = 4-Methoxyphenyl
36: Ar = 4-Methoxyphenyl
Scheme 2.
N
H
ilarly to MS-275, the compounds 31 and 43 also induced
expression of p21 protein in T24 human cancer cell lines
(Fig. 3), and they caused G2/M cell cycle arrest in the
HCT116 human colon cancer cell line (Fig. 4), in accor-
dance with results from the literature.14,15
MeO
Scheme 3.
activity against HDAC-1 (3 lM) and good antiprolifer-
ative activity (1 lM). Chain elongation (compounds 8
and 9) or introduction of substituents on the phenyl ring
(compounds 10–15) only slightly modified the activity.
Interchanging the position of the nitrogen (compounds
15 and 16) did little to improve the potency.
HDAC small molecule inhibitors 31 and 43 were evalu-
ated in vivo in several different tumor xenograft models
250
200
To continue this SAR, the aminophenyl cinnamide
series was explored (Table 2). In this series, interchang-
ing the position of the nitrogen (compounds 25 and 26)
resulted in improvement in antiproliferative activity.
The absence or presence of a methoxy substituent and
the substitution pattern (compounds 26–31) showed up
to a 10-fold difference in HDAC-1 activity, with
compound 31 being the most potent. Introduction of a
secondary amine (compound 32) or substituent variety
(compounds 33–35) did not prove to be beneficial.
150
1 uM
100
50
0
5 uM
DMSO
31
43
Figure 3. Induction of p21WAF1/Cip1 transcription by HDAC inhibitors
in Human HCT116 Cancer Cells. HCT116 cells were stably transfected
with a luciferase reporter plasmid driven by the p21 promoter. Cells
were treated with HDAC inhibitors for 16 h before harvest. Cells were
lysed and luciferase activity was analyzed using substrate from
Promega.
To complete the SAR study, the (pyridin-3-yl)acrylam-
ide (Table 1) and the cinnamide series (Table 2) were
compared to the nicotinamide (compounds 36–38) and
the benzamide series (compounds 39–44) (Table 3).
These new compounds 36–44 showed an improved
range of antiproliferative activity from 0.5 to 3 lM.
Compounds 39, 42, and 43 were found to be quite po-
tent; comparable to MS-275 (3) which is currently
undergoing phase 1 clinical trials.13
Cell Cycle Arrest in Human Cancer Cells
40
35
30
25
20
15
10
5
SubG1
G1
S
G2+M
The cytotoxicity of the most potent compounds toward
normal cells was determined by the proliferation of hu-
man normal mammary epithelial cells (HMEC). Com-
pounds 31 (HMEC IC50 = 39 lM) and 43 (HMEC
IC50 = 15 lM) proved to have the lowest cytotoxicity.
0
DMSO MS-275 Cpd 31 Cpd 31 Cpd 43 Cpd 43
(1uM) (1 uM) (10 uM) (1 uM) (10 uM)
Figure 4. HDAC Inhibitors Caused G2/M arrest of Human Cancer
Cells. Human HCT116 cancer cells were treated with in-house HDAC
inhibitors or MS-275 in 1% DMSO for 16 h before harvesting by
trypsinization. Propidium iodide-stained DNA contents in fixed cells
were analyzed by flow cytometry. The (G2+M)/S ratio was calculated
using the percentage of cells among total gated cells in G2/M phase
divided by the percentage of cells in S phase. Percentages of cells in G1
phase were not affected by HDAC inhibitors.
To confirm the ability of these compounds to inhibit
HDAC in whole cells, they were evaluated for their abil-
ity to induce histone acetylation in T24 human bladder
cancer cells. The anilides 31 and 43 caused dose-depen-
dent histone hyperacetylation in these cells (Fig. 2). Sim-

4052
O. Moradei et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4048–4052
in mice. These models represent the major forms of hu-
man cancers: A549 (non-small cell lung), PANC-1 (pan-
creatic), and SW48 and HCT116 (colon). Compounds
were administered daily by either intraperitoneal injec-
tion (ip) or orally (po). Both compounds showed good
antitumor activity (% of tumor growth inhibitions rela-
tive to vehicle controls, %TGI). Compound 31 showed
up to 73% in SW48 and up to 80% in HCT116 when
dosed at 25 and 20 mg/kg per day (ip route) for 3 weeks.
Compound 43 showed up to 73% in A549 (ip, 40 mg/
kg), up to 84% in PANC1 (po, 50 mg/kg), up to 66%
and 86% in HCT116 (ip, 50 and 75 mg/kg), and up to
66% and 100% in SW48 (ip, 50 and 75 mg/kg). This
activity was obtained without any associated body
weight loss as a measure of gross toxicity. Moreover,
the activity was comparable or better than that of MS-
275 (3) (%TGI: 56, 68, and 46 for HCT116, SW48,
and A549-bearing mice, respectively, when dosed at
20 mg/kg ip) that was used as a positive control in these
experiments.
References and notes
1. Rundlett, S. E.; Carmen, A. A.; Kobayashi, R.; Bavykin,
S.; Turner, B. M.; Grunstein, M. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 14503.
2. (a) Hassig, C. A.; Schreiber, S. L. Curr. Opin. Chem. Biol.
1997, 1, 300; (b) Kouzarides, T. Curr. Opin. Genet. Dev.
1999, 9, 40; Review (c) Strahl, B. D.; Allis, C. D. Nature
2000, 403, 41.
3. Lin, R. J.; Nagy, L.; Inoue, S.; Shao, W.; Miller, W. H.;
Evans, R. M. Nature 1998, 391, 811.
4. (a) Beppu, T.; Iwamoto, Y.; Yoshida, M. U.S. Patent
4,690,918, 1987; Chem. Abstr. 1987, 106, 9389; (b)
Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. J. Biol.
Chem. 1990, 265, 17174; (c) Fleming, I.; Iqbal, J.; Krebs,
E. P. Tetrahedron 1983, 39, 841; (d) Mori, K.; Koseki, K.
Tetrahedron 1988, 44, 6013.
5. (a) Jung, M.; Brosch, G.; Ko¨lle, D.; Scherf, H.; Gerha¨user,
C.; Loidl, P. J. Med. Chem. 1999, 42, 4669; (b) Su, G. H.;
Sohn, T. A.; Ryu, B.; Kern, S. E. Cancer Res. 2000, 60,
3137; (c) Massa, S.; Mai, A.; Sbardella, G.; Esposito, M.;
Ragno, R.; Loidl, P.; Brosch, G. J. Med. Chem. 2001, 44,
2069.
A novel class of HDAC inhibitors, which induce core
histone acetylation in human cancer cells, arylamine ani-
lides (general structure 6), was designed. Active aryl-
amine anilides exhibit in vitro anti-proliferative
activities in various human cancer cells, but not in nor-
mal cells. Selective induction of cell cycle arrest and
apoptosis of human cancer cells in a dose-dependent
manner was observed. At the protein expression level,
compounds 31 and 43 induce p21WAF1/Cip1 expression
and downregulate cyclin A and B1 expression in cancer
cells. Induction of p21WAF1/Cip1 by compounds 31 and
43 starts as early as 8 h, and can last up to 72 h, post-
treatment. It was found that compounds 31 and 43 have
significant antitumor activity in vivo in several mice tu-
mor xenograft models. Compounds 31 and 43 showed
acceptable pharmacokinetic profiles in rats (iv half-life
1.8 and 0.7 h; bioavailability 65% and 20%, respective-
ly). Little toxicity of compounds 31 and 43, compared
to MS-275 (3), was revealed by body weight, white
blood cell counts, and spleen weight measurement.
These results represent a significant step toward the
development of HDAC small molecule inhibitors with
favorable pharmaceutical characteristics.
6. (a) Kim, Y. B.; Lee, K. H.; Sugita, K.; Yoshida, M.;
Horinouchi, S. Oncogene 1999, 18, 2461; (b) Meinke, P.
T.; Liberator, P. Curr. Med. Chem. 2000, 8, 211.
7. Saito, A.; Yamashita, T.; Mariko, Y.; Nosaka, Y.;
Tsuchiya, K.; Ando, T.; Suzuki, T.; Tsuruo, T.; Nakani-
shi, O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4592.
8. Piekarz, R.; Bates, S. Curr. Pharm. Des. 2004, 10, 2289.
9. Lavoie, R.; Bouchain, G.; Frechette, S.; Woo, S. H.;
Abou-Khalil, E.; Leit, S.; Fournel, M.; Yan, P. T.;
Trachy-Bourget, M.-C.; Beaulieu, C.; Li, Z.; Besterman,
J.; Delorme, D. Bioorg. Med. Chem. Lett. 2001, 11, 2847.
10. Bouchain, G.; Leit, S.; Frechette, S.; Abou-Khalil, E.;
Lavoie, R.; Moradei, O.; Woo, S. H.; Fournel, M.; Yan,
P. T.; Kalita, A.; Trachy-Bourget, M.-C.; Beaulieu, C.; Li,
Z.; Robert, M.; MacLeod, R.; Besterman, J.; Delorme, D.
J. Med. Chem. 2003, 46, 820.
11. Full experimental protocols for Schemes 1–3 are described
in Delorme, D.; Woo, S. H.; Vaisburg A.; Moradei, O.
et al. WO 03/024448.
12. Carmen, A. A.; Rundlett, S. E.; Grunstein, M. J. Biol.
Chem. 1996, 271, 15837.
13. (a) Jaboin, J.; Wild, J.; Hamidi, H.; Khanna, C.; Jai Kim,
C.; Robey, R.; Bates, S. E.; Thiele, C. J. Cancer Res. 2002,
32, 6108; (b) Acharya, M.; Sparreboom, A.; Sausville, E.;
Conley, B.; Doroshow, J.; Venitz, J.; Figg, W. Cancer
Chemother. Pharmacol. 2006, 57, 275.
14. Maeda, T.; Nagaoka, Y.; Kawai, Y.; Takagani, N.;
Yasuda, C.; Yogosaga, S.; Sowa, Y.; Sakai, T.; Uesato,
S. Biol. Pharm. Bull. 2005, 28, 849.
Acknowledgment
15. Hirose, T.; Sowa, Y.; Takahashi, S.; Saito, S.; Yasuda, C.;
Shindo, N.; Furuichi, K.; Sakai, T. Oncogene 2003, 22,
7762.
Authors are grateful to Dr. Tammy Mallais for her help
in preparing the manuscript.