4-(Heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides and their analogs as a novel class of histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.12.057

The synthesis and biological evaluation of a variety of 4-(heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides and their analogs is described. Some of these compounds were shown to inhibit HDAC1 with IC₅₀ values below the micromolar range, indu

Full text of DOI:10.1016/j.bmcl.2007.12.057

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1502–1506
4-(Heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides
and their analogs as a novel class of histone deacetylase inhibitors
a,
a
a,
Sylvie Frechette, Silvana Leit, Soon Hyung Woo, Guillaume Lapointe,a
*
´
Guillaume Jeannotte,a Oscar Moradei,a Isabelle Paquin,a Giliane Bouchain,a
a
a
a
a
´
´ ´
Stephane Raeppel, Frederic Gaudette, Nancy Zhou, Arkadii Vaisburg,
Marielle Fournel,b Pu Theresa Yan,b Marie-Claude Trachy-Bourget,b Ann Kalita,b
Marie-France Robert,b Aihua Lu,b Jubrail Rahil,b A. Robert MacLeod,b,à
Jeffrey M. Besterman,b Zuomei Lib and Daniel Delormea,§
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 26 October 2007; revised 18 December 2007; accepted 21 December 2007
Available online 25 December 2007
Abstract—The synthesis and biological evaluation of a variety of 4-(heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides and
their analogs is described. Some of these compounds were shown to inhibit HDAC1 with IC50 values below the micromolar range,
induce hyperacetylation of histones, upregulate expression of the tumor suppressor p21WAF1/Cip1, and inhibit proliferation of human
cancer cells. In addition, certain compounds of this class were active in several human tumor xenograft models in vivo.
Ó 2008 Elsevier Ltd. All rights reserved.
Histone deacetylases (HDACs) catalyze the hydrolysis of
acetyl groups on the NH2-terminal lysine residues of the
core nucleosomal histones.1 The acetylation status of
the core histones correlates with transcriptional activity
of certain genes. HDAC activity is generally associated
with transcriptional repression. Abnormally increased
HDAC activity has been associated with the development
of certain human cancers.2 In recent years, inhibition of
HDACs has emerged as a potential strategy to reverse
aberrant epigenetic changes associated with cancer.3
Small molecules with hydroxamic acid functional groups
such as natural product trichostatin A (TSA)4 (1) and it’s
analogues,5 suberoylanilide hydroxamic acid (SAHA,
ZolinzaTM, Merck & Co., Inc.)6 (2) and synthetic com-
pounds such as the 2-aminoanilide MS-2757 (3) and our
isotype specific, oral product candidate MGCD0103,8
are potent HDAC inhibitors (Fig. 1). Some of these com-
pounds demonstrate in vivo anti-tumor activity and are
currently under clinical evaluation and SAHA, has re-
cently been approved for the treatment of advanced cuta-
neous T-cell lymphoma.
In the course of searching for novel HDAC inhibitors
withhigh potency andgoodsafetyprofiles, we recently de-
signed 4-[(s-triazin-2-ylamino)methyl]-N-(2-aminophe-
nyl)benzamides (4).9 As a further development of
HDAC inhibitors with better pharmaceutical and phar-
macokinetic properties, we have synthesized 4-(hetero-
arylaminomethyl)-N-(2-aminophenyl)benzamides
(5)
bearing a 5-membered heteroaromatic ring which showed
significant improvement in anti-tumor activities both
in vitro and in vivo. The structure–activity relationships
(SAR), the anti-proliferative activity and the in vivo effi-
cacy of these novel HDAC inhibitors will be discussed.
Keywords: Histone deacetylase inhibitors; HDAC; Benzamides; MG-
CD0103; Anticancer agents.
*
Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337
0550; e-mail: frechettes@methylgene.com
Present address: Auspex Pharmaceuticals, 1261 Liberty Way, Vista,
CA 92081-8356, USA.
à
Present address: Takeda San Diego, 10410 Science Center Drive, San
Diego, CA 92121, USA.
The first series of compounds bearing a 5-membered
heteroaromatic ring 6–12 (Table 1) was synthesized
using two different approaches (Scheme 1). To generate
§
Present address: Neurochem Inc., 275 Armand-Frappier Blvd.,
Laval, QC, Canada H7V 4A7.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.057

´
S. Frechette et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1502–1506
1503
O
O
O
O
H
O
N
H
NH2
NHOH
N
H
NHOH
N
N
O
N
TSA (1)
SAHA (2)
MS-275 (3)
O
NR3R4
N
O
N
N
N
N
NH2
N
R1R2N
N
N
H
NH2
H
H
H
N
X
NH2
H
N
N
N
O
Y
Z
MGCD0103
4
5
O
Figure 1. Small molecule HDAC inhibitors.
Table 1. In vitro activities of compounds 6–12
1H-imidazole-2-thiol was introduced via a Mitsunobu
reaction. The Boc-group deprotection with TFA affor-
ded compound 6. Compounds 7 and 10 were synthesized
in a similar fashion. To generate the heteroarylamino
derivatives a reductive amination between methyl 4-
formylbenzoate and 5-bromothiazol-2-amine was used
to generate compound 14. The ester functionality was
hydrolyzed and then coupled with 1,2-phenylenediamine
using BOP§ as a coupling agent to afford final product 8.
Compounds 9, 11 and 12 were obtained similarly to
compound 8.
X
NH2
H
N
O
Compound
X
HDAC1a MTT
IC50
(lM)
HCT116
b
IC50 (lM)
H
N
6
7
0.5
5
S
S
The second series was based on benzo-fused heteroaro-
matic systems (compounds 15–25, Table 2). The reac-
tion between 6-aminobenzothiazole-2-thiol and methyl
4-(bromomethyl)benzoate afforded compound 26
(Scheme 2). The amino group of this material was alkyl-
ated with 3-(bromomethyl)pyridine; the ester functional-
ity was hydrolyzed to the corresponding acid which was
then coupled with 1,2-phenylenediamine using BOP§ as
a coupling agent, to afford final product 22. Other ben-
zothiazol-2-ylthio derivatives (compounds 15, 23 and
24) and benzimidazol-2-ylthio derivatives (compounds
16 and 21) were synthesized using the same approach.
A reductive amination between 5-bromo-benzothiazol-
2-amine and methyl 4-formylbenzoate generated com-
pound 27. This intermediate was hydrolyzed and the
corresponding intermediate acid was coupled with 1,2-
phenylenediamine to generate the final compound 19.
Compounds 17, 18 and 20 were synthesized similarly
to compound 19. Compound 25 was obtained via a
reductive amination followed by a Mitsunobu reaction
on intermediate 28 and then completed the same way
as the other compounds.
N
N
N
1
5
Br
S
8
0.4
0.2
0.08
0.07
0.3
0.4
0.6
0.4
0.3
0.3
NH
N
HN N
N
9
S
N
H
Ph
O
10
11
12
S
N
N
Ph
NH
HN
N
In our HDAC oncology program, we targeted HDAC1
in our design strategy since our work and that of others
have clearly linked inhibition of this enzyme with his-
tone hyperacetylation and inhibition of cell prolifera-
tion.10 Thus, the series of compounds presented here
are potent HDAC1 inhibitors. As Tables 1 and 2 show,
the IC50 values range from 30 nM to 1 lM, when tested
H
N
S
Cl
N
a Inhibition of recombinant HDAC1.
b Cytotoxicity/proliferation of human cancer HCT116 cells.
the heteroarylthio derivatives, 1,2-phenylenediamine
was monoprotected with a Boc-group and coupled with
4-(methoxycarbonyl)benzoic acid to generate amide 13.
The ester functionality was reduced to the alcohol and
§
Abbreviations: BOP, (benzotriazol-1-yloxy)tris (dimethyl-amino)-
phosphoniumhexafluorophosphate; AMC, aminomethylcoumarin;
MTT, 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide.

´
S. Frechette et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1502–1506
1504
O
O
1. Boc2O, DMAP, THF
1. DIBAL-H, THF
N
H
N
H
H2N
2. 4-(Methoxycarbonyl)-
benzoic acid, EDC,
HOBt, Et3N, DMF
2. Het-SH, DEAD, Ph3P, THF
3. TFA, DCM
NHBoc
S
NH2
NH2
MeO2C
Het
13
6 Het = 1
H
-imidazol-2-yl
1. LiOH, H2O, THF
2. 1,2-Phenylenediamine
BOP, Et3N, DMF
O
O
O
Het-NH
OMe
N
H
H
N
NaHB(OAc)3
THF
Het NH
OMe
NH2
OHC
Het
14 Het = 5-bromothiazol-2-yl
8
Het = 5-bromothiazol-2-yl
Scheme 1.
Table 2. In vitro activities of compounds 15–25
X
NH2
H
N
O
Compound
X
HDAC1a
MTT HCT116b
MTT HMECb
H4-Acc
IC50 (lM)
IC50 (lM)
IC50 (lM)
EC50 (lM)
S
N
15
16
0.1
0.8
0.3
3
3
S
S
H
N
0.09
0.1
14
<1
N
H
N
17
18
19
0.3
0.2
0.4
27
17
6
1
1
2
NH
N
O
NH
NH
0.1
N
Cl
N
S
0.05
Br
MeO
S
N
20
21
22
23
0.03
0.07
0.8
0.2
0.2
1
5
10
1
1
2
3
NH
H
N
MeO
S
N
S
N
N
H
S
S
>50
N
S
N
N
H
0.07
1
8
N

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S. Frechette et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1502–1506
1505
Table 2 (continued)
Compound
X
HDAC1a
MTT HCT116b
MTT HMECb
H4-Acc
IC50 (lM)
IC50 (lM)
IC50 (lM)
EC50 (lM)
H
N
S
N
N
N
24
25
0.07
0.06
0.2
0.2
15
15
5
S
O
O
O
S
N
1
NH
a Inhibition of recombinant HDAC1.
b Cytotoxicity/proliferation in human cancer HCT116 cells and human normal mammary epithelial (HMEC) cells.
c Relative effective concentration of compounds in induction of histone H4 acetylation in T24 human cancer cells, relative to MS-275 at 1 lM.
Human T24 cells were treated with compounds at 0, 1, 5, 25 lM for 16 h. Cells were harvested and histones were acid-extracted. Histones were
analyzed by SDS–PAGE and immunoblotting with antibodies specific for either H4 histones or acetylated H4 histones.
1. 3-(Bromomethyl)pyridine
Methyl 4-(bromomethyl)-
Et3N, K2CO3, NaI
benzoate
DMF
H
O
O
N
N
H2N
2. LiOH, H2O, THF
S
N
S
N
H2N
S
N
SH
S
HN
S
OMe
3. 1,2-Phenylenediamine
BOP, Et3N, DMF
H2N
26
22
1. LiOH, H2O, THF
2. 1,2-Phenylenediamine
BOP, Et3N, DMF
H2N
HN
Methyl 4-formylbenzoate
NaHB(OAc)3, THF
Br
S
Br
Br
S
S
N
OMe
O
NH
NH2
NH
N
N
27
O
19
1. 2-Morpholinoethanol, DEAD
PPh3, THF, 0oC to rt
O
Methyl 4-formylbenzoate
Bu2SnCl2, PhSiH3, THF HO
O
HO
S
N
S
N
CO2Me
2. LiOH, THF, H2O
N
S
N
NH
25
HN
NH2
NH
O
3. 1,2-Phenylenediamine
BOP, Et3N, DMF
28
H2N
Scheme 2.
§
using BocLys(acetyl)AMC,11 as a substrate. Also
shown are data demonstrating the in vitro anti-prolifer-
ative potency of these compounds in the human colon
cancer cell line, HCT116 (measured using the MTT§ re-
agent). A desirable selectivity, ranging from fourfold to
greater than 50-fold in potency, is observed in the anti-
proliferative activity in cancer cells as compared to nor-
mal human mammary epithelial cells (HMEC) (Table
2).
and 23 bearing bulky aromatic substituents were weaker
in inhibiting HCT116 cell proliferation (1 lM) com-
pared to compounds 15–21 which showed sub-micromo-
lar activities in the same assay. Interestingly, compounds
24 and 25 with a large non-aromatic substituent were as
potent as compounds 15–21 in both assays. Consistent
with inhibiting cellular HDACs, these inhibitors induced
histone hyperacetylation in T24 human cancer cells
(EC50 1–5 lM, Table 2).
The imidazole derivatives (compounds 6 and 7, Table 1)
showed reasonable potency against HDAC1 but low
anti-proliferative activity (5 lM). The introduction of
a non-polar substituent on the 5-membered ring hetero-
cycle (compounds 8–12) improved anti-proliferative
activity (0.3–0.6 lM) while maintaining high enzymatic
inhibitory potency (0.07–0.40 lM).
Also consistent with their mechanism of action, such
inhibitors induced the expression of the cyclin-depen-
dent kinase inhibitor p21WAF1/Cip1 (EC50 ꢀ 2 lM) in
the T24 human cancer cell line. For example, com-
pounds 20, 21, and 25 gave EC50s of 0.8, 0.6, and
1.0 lM, respectively. Furthermore, all three compounds
caused G2/M cell cycle arrest in the HCT116 human co-
lon cancer cell line (Fig. 2, data shown only for 25).10
To further explore the SAR of heteroarylaminomethyl-
based HDAC inhibitors we have designed and synthe-
sized analogues bearing bicyclic annealated heteroaro-
matic systems 15–25 (Table 2). These compounds
(regardless of the nature of the heteroaromatic ring or
substitution pattern) exhibited sub-micromolar HDAC1
inhibitory activities and they also discriminated between
cancer cells (exemplified by the HCT116 colon cancer
cell line) and normal cells (Table 2). Compounds 22
Compounds 20, 21, and 25 were evaluated in vivo in sev-
eral different human tumor xenograft models in mice
representing the major forms of human cancers:
PANC-1 (pancreatic), SW48 (colon), COLO205 (colon)
and A549 (lung). Compounds were administered daily
for 14 days by either intraperitoneal (ip) injection or or-
ally (po). Compound 25 showed the best anti-tumor
activity quantified as % of tumor growth relative to vehi-

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G2
neg ctl
1%
MS-275 MS-275 25 (1uM)
25
(10uM)
DMSO (1uM) (10uM)
Figure 2. HDAC inhibitors caused G2/M arrest of human cancer cells.
Human HCT116 cancer cells were treated with compound 25 or MS-
275 in 1% DMSO for 16 h before harvesting by trypsinization.
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Table 3. In vivo anti-tumor activity of compound 25
Dose (mg/kg)
% Tumor growth inhibition
20 (ip)
90 (po)
74% (SW48), 65% (A549), 70% (PANC-1)
72% (SW48), 65% (A549), 62% (COLO205)
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cle treated controls, (Table 3, also see supplementary
material for the anti-tumor effect of compound 25 com-
pared to MS-275 in PANC-1 Human Pancreatic Carci-
noma Model).
This in vivo anti-tumor activity was obtained without
any associated body weight loss as a measure of gross
toxicity. We designed and synthesized a novel class of
HDAC inhibitors, the 4-(heteroarylaminomethyl)-N-(2-
aminophenyl)-benzamides and their analogues. These
compounds exhibited in vitro anti-proliferative activities
in numerous human cancer cells with little or no activity
on normal cells, induced p21WAF1/Cip1 expression and in-
duced hyperacetylation of core histones. They also
caused cell cycle arrest of human cancer cells in a
dose-dependent manner. The most promising HDAC
inhibitors of the 4-(heteroarylamino/thiomethyl)-N-(2-
aminophenyl)-benzamide series were found to be com-
pounds 20, 21 and 25. These compounds have attractive
in vitro profiles and show significant anti-tumor activity
in vivo in several human cancer xenograft model mod-
els. These results represent a significant step towards
the development of small molecule HDAC inhibitors
with favorable pharmaceutical properties.
´
8. Zhou, N.; Moradei, O.; Raeppel, S.; Leit, S.; Frechette,
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Bourget, M.; Yan, P. T.; Liu, J.; Li, Z.; Rahil, G.;
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(HDAC) inhibitors: synthesis and antiproliferative eval-
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Supplementary data
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.12.057.
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